Ectopic norrin induces growth of ocular capillaries and restores normal retinal angiogenesis in Norrie disease mutant mice.

PubMed

Ohlmann, Andreas; Scholz, Michael; Goldwich, Andreas; Chauhan, Bharesh K; Hudl, Kristiane; Ohlmann, Anne V; Zrenner, Eberhart; Berger, Wolfgang; Cvekl, Ales; Seeliger, Mathias W; Tamm, Ernst R

2005-02-16

Norrie disease is an X-linked retinal dysplasia that presents with congenital blindness, sensorineural deafness, and mental retardation. Norrin, the protein product of the Norrie disease gene (NDP), is a secreted protein of unknown biochemical function. Norrie disease (Ndp(y/-)) mutant mice that are deficient in norrin develop blindness, show a distinct failure in retinal angiogenesis, and completely lack the deep capillary layers of the retina. We show here that the transgenic expression of ectopic norrin under control of a lens-specific promoter restores the formation of a normal retinal vascular network in Ndp(y/-) mutant mice. The improvement in structure correlates with restoration of neuronal function in the retina. In addition, lenses of transgenic mice with ectopic expression of norrin show significantly more capillaries in the hyaloid vasculature that surrounds the lens during development. In vitro, lenses of transgenic mice in coculture with microvascular endothelial cells induce proliferation of the cells. Transgenic mice with ectopic expression of norrin show more bromodeoxyuridine-labeled retinal progenitor cells at embryonic day 14.5 and thicker retinas at postnatal life than wild-type littermates, indicating a putative direct neurotrophic effect of norrin. These data provide direct evidence that norrin induces growth of ocular capillaries and that pharmacologic modulation of norrin might be used for treatment of the vascular abnormalities associated with Norrie disease or other vascular disorders of the retina.

ELAV, a Drosophila neuron-specific protein, mediates the generation of an alternatively spliced neural protein isoform.

PubMed

Koushika, S P; Lisbin, M J; White, K

1996-12-01

Tissue-specific alternative pre-mRNA splicing is a widely used mechanism for gene regulation and the generation of different protein isoforms, but relatively little is known about the factors and mechanisms that mediate this process. Tissue-specific RNA-binding proteins could mediate alternative pre-mRNA splicing. In Drosophila melanogaster, the RNA-binding protein encoded by the elav (embryonic lethal abnormal visual system) gene is a candidate for such a role. The ELAV protein is expressed exclusively in neurons, and is important for the formation and maintenance of the nervous system. In this study, photoreceptor neurons genetically depleted of ELAV, and elav-null central nervous system neurons, were analyzed immunocytochemically for the expression of neural proteins. In both situations, the lack of ELAV corresponded with a decrease in the immunohistochemical signal of the neural-specific isoform of Neuroglian, which is generated by alternative splicing. Furthermore, when ELAV was expressed ectopically in cells that normally express only the non-neural isoform of Neuroglian, we observed the generation of the neural isoform of Neuroglian. Drosophila ELAV promotes the generation of the neuron-specific isoform of Neuroglian by the regulation of pre-mRNA splicing. The findings reported in this paper demonstrate that ELAV is necessary, and the ectopic expression of ELAV in imaginal disc cells is sufficient, to mediate neuron-specific alternative splicing.

Clustered metallothionein genes are co-regulated in rice and ectopic expression of OsMT1e-P confers multiple abiotic stress tolerance in tobacco via ROS scavenging

PubMed Central

2012-01-01

Background Metallothioneins (MT) are low molecular weight, cysteine rich metal binding proteins, found across genera and species, but their function(s) in abiotic stress tolerance are not well documented. Results We have characterized a rice MT gene, OsMT1e-P, isolated from a subtractive library generated from a stressed salinity tolerant rice genotype, Pokkali. Bioinformatics analysis of the rice genome sequence revealed that this gene belongs to a multigenic family, which consists of 13 genes with 15 protein products. OsMT1e-P is located on chromosome XI, away from the majority of other type I genes that are clustered on chromosome XII. Various members of this MT gene cluster showed a tight co-regulation pattern under several abiotic stresses. Sequence analysis revealed the presence of conserved cysteine residues in OsMT1e-P protein. Salinity stress was found to regulate the transcript abundance of OsMT1e-P in a developmental and organ specific manner. Using transgenic approach, we found a positive correlation between ectopic expression of OsMT1e-P and stress tolerance. Our experiments further suggest ROS scavenging to be the possible mechanism for multiple stress tolerance conferred by OsMT1e-P. Conclusion We present an overview of MTs, describing their gene structure, genome localization and expression patterns under salinity and development in rice. We have found that ectopic expression of OsMT1e-P enhances tolerance towards multiple abiotic stresses in transgenic tobacco and the resultant plants could survive and set viable seeds under saline conditions. Taken together, the experiments presented here have indicated that ectopic expression of OsMT1e-P protects against oxidative stress primarily through efficient scavenging of reactive oxygen species. PMID:22780875

Clustered metallothionein genes are co-regulated in rice and ectopic expression of OsMT1e-P confers multiple abiotic stress tolerance in tobacco via ROS scavenging.

PubMed

Kumar, Gautam; Kushwaha, Hemant Ritturaj; Panjabi-Sabharwal, Vaishali; Kumari, Sumita; Joshi, Rohit; Karan, Ratna; Mittal, Shweta; Pareek, Sneh L Singla; Pareek, Ashwani

2012-07-10

Metallothioneins (MT) are low molecular weight, cysteine rich metal binding proteins, found across genera and species, but their function(s) in abiotic stress tolerance are not well documented. We have characterized a rice MT gene, OsMT1e-P, isolated from a subtractive library generated from a stressed salinity tolerant rice genotype, Pokkali. Bioinformatics analysis of the rice genome sequence revealed that this gene belongs to a multigenic family, which consists of 13 genes with 15 protein products. OsMT1e-P is located on chromosome XI, away from the majority of other type I genes that are clustered on chromosome XII. Various members of this MT gene cluster showed a tight co-regulation pattern under several abiotic stresses. Sequence analysis revealed the presence of conserved cysteine residues in OsMT1e-P protein. Salinity stress was found to regulate the transcript abundance of OsMT1e-P in a developmental and organ specific manner. Using transgenic approach, we found a positive correlation between ectopic expression of OsMT1e-P and stress tolerance. Our experiments further suggest ROS scavenging to be the possible mechanism for multiple stress tolerance conferred by OsMT1e-P. We present an overview of MTs, describing their gene structure, genome localization and expression patterns under salinity and development in rice. We have found that ectopic expression of OsMT1e-P enhances tolerance towards multiple abiotic stresses in transgenic tobacco and the resultant plants could survive and set viable seeds under saline conditions. Taken together, the experiments presented here have indicated that ectopic expression of OsMT1e-P protects against oxidative stress primarily through efficient scavenging of reactive oxygen species.

Small Heat Shock Proteins Can Release Light Dependence of Tobacco Seed during Germination1[OPEN

PubMed Central

Koo, Hyun Jo; Park, Soo Min; Kim, Keun Pill; Suh, Mi Chung; Lee, Mi Ok; Lee, Seong-Kon; Xinli, Xia

2015-01-01

Small heat shock proteins (sHSPs) function as ATP-independent molecular chaperones, and although the production and function of sHSPs have often been described under heat stress, the expression and function of sHSPs in fundamental developmental processes, such as pollen and seed development, have also been confirmed. Seed germination involves the breaking of dormancy and the resumption of embryo growth that accompany global changes in transcription, translation, and metabolism. In many plants, germination is triggered simply by imbibition of water; however, different seeds require different conditions in addition to water. For small-seeded plants, like Arabidopsis (Arabidopsis thaliana), lettuce (Lactuca sativa), tomato (Solanum lycopersicum), and tobacco (Nicotiana tabacum), light is an important regulator of seed germination. The facts that sHSPs accumulate during seed development, sHSPs interact with various client proteins, and seed germination accompanies synthesis and/or activation of diverse proteins led us to investigate the role of sHSPs in seed germination, especially in the context of light dependence. In this study, we have built transgenic tobacco plants that ectopically express sHSP, and the effect was germination of the seeds in the dark. Administering heat shock to the seeds also resulted in the alleviation of light dependence during seed germination. Subcellular localization of ectopically expressed sHSP was mainly observed in the cytoplasm, whereas heat shock-induced sHSPs were transported to the nucleus. We hypothesize that ectopically expressed sHSPs in the cytoplasm led the status of cytoplasmic proteins involved in seed germination to function during germination without additional stimulus and that heat shock can be another signal that induces seed germination. PMID:25604531

Ectopic Expression of Nolz-1 in Neural Progenitors Promotes Cell Cycle Exit/Premature Neuronal Differentiation Accompanying with Abnormal Apoptosis in the Developing Mouse Telencephalon

PubMed Central

Chang, Sunny Li-Yun; Chen, Shih-Yun; Huang, Huai-Huei; Ko, Hsin-An; Liu, Pei-Tsen; Liu, Ya-Chi; Chen, Ping-Hau; Liu, Fu-Chin

2013-01-01

Nolz-1, as a murine member of the NET zinc-finger protein family, is expressed in post-mitotic differentiating neurons of striatum during development. To explore the function of Nolz-1 in regulating the neurogenesis of forebrain, we studied the effects of ectopic expression of Nolz-1 in neural progenitors. We generated the Cre-loxP dependent conditional transgenic mice in which Nolz-1 was ectopically expressed in proliferative neural progenitors. Ectopic expression of Nolz-1 in neural progenitors by intercrossing the Nolz-1 conditional transgenic mice with the nestin-Cre mice resulted in hypoplasia of telencephalon in double transgenic mice. Decreased proliferation of neural progenitor cells were found in the telencephalon, as evidenced by the reduction of BrdU−, Ki67− and phospho-histone 3-positive cells in E11.5–12.5 germinal zone of telencephalon. Transgenic Nolz-1 also promoted cell cycle exit and as a consequence might facilitate premature differentiation of progenitors, because TuJ1-positive neurons were ectopically found in the ventricular zone and there was a general increase of TuJ1 immunoreactivity in the telencephalon. Moreover, clusters of strong TuJ1-expressing neurons were present in E12.5 germinal zone. Some of these strong TuJ1-positive clusters, however, contained apoptotic condensed DNA, suggesting that inappropriate premature differentiation may lead to abnormal apoptosis in some progenitor cells. Consistent with the transgenic mouse analysis in vivo, similar effects of Nozl-1 over-expression in induction of apoptosis, inhibition of cell proliferation and promotion of neuronal differentiation were also observed in three different N18, ST14A and N2A neural cell lines in vitro. Taken together, our study indicates that ectopic expression of Nolz-1 in neural progenitors promotes cell cycle exit/premature neuronal differentiation and induces abnormal apoptosis in the developing telencephalon. PMID:24073229

Ectopic expression of nolz-1 in neural progenitors promotes cell cycle exit/premature neuronal differentiation accompanying with abnormal apoptosis in the developing mouse telencephalon.

PubMed

Chang, Sunny Li-Yun; Chen, Shih-Yun; Huang, Huai-Huei; Ko, Hsin-An; Liu, Pei-Tsen; Liu, Ya-Chi; Chen, Ping-Hau; Liu, Fu-Chin

2013-01-01

Nolz-1, as a murine member of the NET zinc-finger protein family, is expressed in post-mitotic differentiating neurons of striatum during development. To explore the function of Nolz-1 in regulating the neurogenesis of forebrain, we studied the effects of ectopic expression of Nolz-1 in neural progenitors. We generated the Cre-loxP dependent conditional transgenic mice in which Nolz-1 was ectopically expressed in proliferative neural progenitors. Ectopic expression of Nolz-1 in neural progenitors by intercrossing the Nolz-1 conditional transgenic mice with the nestin-Cre mice resulted in hypoplasia of telencephalon in double transgenic mice. Decreased proliferation of neural progenitor cells were found in the telencephalon, as evidenced by the reduction of BrdU-, Ki67- and phospho-histone 3-positive cells in E11.5-12.5 germinal zone of telencephalon. Transgenic Nolz-1 also promoted cell cycle exit and as a consequence might facilitate premature differentiation of progenitors, because TuJ1-positive neurons were ectopically found in the ventricular zone and there was a general increase of TuJ1 immunoreactivity in the telencephalon. Moreover, clusters of strong TuJ1-expressing neurons were present in E12.5 germinal zone. Some of these strong TuJ1-positive clusters, however, contained apoptotic condensed DNA, suggesting that inappropriate premature differentiation may lead to abnormal apoptosis in some progenitor cells. Consistent with the transgenic mouse analysis in vivo, similar effects of Nozl-1 over-expression in induction of apoptosis, inhibition of cell proliferation and promotion of neuronal differentiation were also observed in three different N18, ST14A and N2A neural cell lines in vitro. Taken together, our study indicates that ectopic expression of Nolz-1 in neural progenitors promotes cell cycle exit/premature neuronal differentiation and induces abnormal apoptosis in the developing telencephalon.

Endometrial seedlings. A survival instinct? Immunomodulation and its role in the pathophysiology of endometriosis.

PubMed

Portelli, M; Pollacco, J; Sacco, K; Schembri-Wismayer, P; Calleja-Agius, J

2011-12-01

Endometriosis occurs when ectopic cells from the endometrium implant within the peritoneum. It is considered as a disease of multifactorial aetiology and affects 7-10% of women of reproductive age worldwide. In endometriosis, the immune system is thought to be dysfunctional and various studies have shown cytokine imbalance. Commonly upregulated cytokines include Tumour necrosis factor-alpha, interferon gamma and interleukin-10. Through analysis of the molecular makeup of the peritoneal fluid, a change is shown to occur, conferring resistance from macrophages and lymphocytes to endometrial cells. This is possibly due to a reduced Inter-cellular adhesion molecule-1 synthesis. Survival of ectopic endometrial cells also arises through the expression of human leukocyte antigens. Apart from the survival of ectopic/eutopic cells in endometriosis, there is marked cellular proliferation, which has also been attributed to a change in the expression of proteins such as Bcl-2-Associated X protein, B-cell lymphoma-2 protein, transforming growth factor-beta and the enzyme aromatase. Danazol and aromatase inhibitors modulate the immune system, thus allowing partial restoration of cytokine levels. Pharmacogenomics may be the way forward in developing novel treatment modalities for endometriosis.

The tripartite leader sequence is required for ectopic expression of HAdV-B and HAdV-E E3 CR1 genes.

PubMed

Bair, Camden R; Kotha Lakshmi Narayan, Poornima; Kajon, Adriana E

2017-05-01

The unique repertoire of genes that characterizes the early region 3 (E3) of the different species of human adenovirus (HAdV) likely contributes to their distinct pathogenic traits. The function of many E3 CR1 proteins remains unknown possibly due to unidentified intrinsic properties that make them difficult to express ectopically. This study shows that the species HAdV-B- and HAdV-E-specific E3 CR1 genes can be expressed from vectors carrying the HAdV tripartite leader (TPL) sequence but not from traditional mammalian expression vectors. Insertion of the TPL sequence upstream of the HAdV-B and HAdV-E E3 CR1 open reading frames was sufficient to rescue protein expression from pCI-neo constructs in transfected 293T cells. The detection of higher levels of HAdV-B and HAdV-E E3 CR1 transcripts suggests that the TPL sequence may enhance gene expression at both the transcriptional and translational levels. Our findings will facilitate the characterization of additional AdV E3 proteins. Copyright © 2017 Elsevier Inc. All rights reserved.

Adalimumab Treatment in Biologically Naïve Crohn's Disease: Relationship with Ectopic MUC5AC Expression and Endoscopic Improvement

PubMed Central

Mizoshita, Tsutomu; Tanida, Satoshi; Tsukamoto, Hironobu; Ozeki, Keiji; Katano, Takahito; Nishiwaki, Hirotaka; Ebi, Masahide; Mori, Yoshinori; Kubota, Eiji; Kataoka, Hiromi; Kamiya, Takeshi; Joh, Takashi

2014-01-01

Background. Adalimumab (ADA) is effective for patients with Crohn's disease (CD). However, there have been few reports on ADA therapy with respect to its relationship with pathologic findings and drug efficacy in biologically naïve CD cases. Methods. Fifteen patients with active biologically naïve CD were treated with ADA. We examined them clinically and pathologically with ectopic MUC5AC expression in the lesions before and after 12 and 52 weeks of ADA therapy, retrospectively. Results. Both mean CD activity index scores and serum C-reactive protein values were significantly lower after ADA therapy (P < 0.001). In the MUC5AC negative group, all cases exhibited clinical remission (CR) and endoscopic improvement at 52 weeks. In MUC5AC positive groups, loss of MUC5AC expression was detected in cases having CR and endoscopic improvement at 52 weeks, while remnant ectopic MUC5AC expression was observed in those exhibiting no endoscopic improvement and flare up after 52 weeks. Conclusions. ADA leads to CR and endoscopic improvement in biologically naïve CD cases. In addition, ectopic MUC5AC expression may be a predictive marker of flare up and endoscopic improvement in the intestines of CD patients. PMID:24829572

FGFR1 inhibits skeletal muscle atrophy associated with hindlimb suspension

PubMed Central

Eash, John; Olsen, Aaron; Breur, Gert; Gerrard, Dave; Hannon, Kevin

2007-01-01

Background Skeletal muscle atrophy can occur under many different conditions, including prolonged disuse or immobilization, cachexia, cushingoid conditions, secondary to surgery, or with advanced age. The mechanisms by which unloading of muscle is sensed and translated into signals controlling tissue reduction remains a major question in the field of musculoskeletal research. While the fibroblast growth factors (FGFs) and their receptors are synthesized by, and intimately involved in, embryonic skeletal muscle growth and repair, their role maintaining adult muscle status has not been examined. Methods We examined the effects of ectopic expression of FGFR1 during disuse-mediated skeletal muscle atrophy, utilizing hindlimb suspension and DNA electroporation in mice. Results We found skeletal muscle FGF4 and FGFR1 mRNA expression to be modified by hind limb suspension,. In addition, we found FGFR1 protein localized in muscle fibers within atrophying mouse muscle which appeared to be resistant to atrophy. Electroporation and ectopic expression of FGFR1 significantly inhibited the decrease in muscle fiber area within skeletal muscles of mice undergoing suspension induced muscle atrophy. Ectopic FGFR1 expression in muscle also significantly stimulated protein synthesis in muscle fibers, and increased protein degradation in weight bearing muscle fibers. Conclusion These results support the theory that FGF signaling can play a role in regulation of postnatal skeletal muscle maintenance, and could offer potentially novel and efficient therapeutic options for attenuating muscle atrophy during aging, illness and spaceflight. PMID:17425786

MicroRNA203a suppresses glioma tumorigenesis through an ATM-dependent interferon response pathway

PubMed Central

Yang, Chuan He; Wang, Yinan; Sims, Michelle; Cai, Chun; He, Ping; Häcker, Hans; Yue, Junming; Cheng, Jinjun; Boop, Frederick A.; Pfeffer, Lawrence M.

2017-01-01

Glioblastoma (GBM) is a deadly and incurable brain tumor. Although microRNAs (miRNAs) play critical roles in regulating the cancer cell phenotype, the underlying mechanisms of how they regulate tumorigenesis are incompletely understood. We previously showed that miR-203a is expressed at relatively low levels in GBM patients, and ectopic miR-203a expression in GBM cell lines inhibited cell proliferation and migration, increased sensitivity to apoptosis induced by interferon (IFN) or temozolomide in vitro, and inhibited GBM tumorigenesis in vivo. Here we show that ectopic expression of miR-203a in GBM cell lines promotes the IFN response pathway as evidenced by increased IFN production and IFN-stimulated gene (ISG) expression, and high basal tyrosine phosphorylation of multiple STAT proteins. Importantly, we identified that miR-203a directly suppressed the protein levels of ataxia-telangiectasia mutated (ATM) kinase that negatively regulates IFN production. We found that high ATM expression in GBM correlates with poor patient survival and that ATM expression is inversely correlated with miR-203a expression. Knockout of ATM expression and inhibition of ATM function in GBM cell lines inhibited cell proliferation and migration, increased sensitivity to apoptosis induced by therapeutic agents in vitro, and markedly suppressed GBM tumor growth and promoted animal survival. In contrast, restoring ATM levels in GBM cells ectopically expressing miR-203a increased tumorigenicity and decreased animal survival. Our study suggests that low miR-203a expression in GBM suppresses the interferon response through an ATM-dependent pathway. PMID:29348882

MicroRNA203a suppresses glioma tumorigenesis through an ATM-dependent interferon response pathway.

PubMed

Yang, Chuan He; Wang, Yinan; Sims, Michelle; Cai, Chun; He, Ping; Häcker, Hans; Yue, Junming; Cheng, Jinjun; Boop, Frederick A; Pfeffer, Lawrence M

2017-12-22

Glioblastoma (GBM) is a deadly and incurable brain tumor. Although microRNAs (miRNAs) play critical roles in regulating the cancer cell phenotype, the underlying mechanisms of how they regulate tumorigenesis are incompletely understood. We previously showed that miR-203a is expressed at relatively low levels in GBM patients, and ectopic miR-203a expression in GBM cell lines inhibited cell proliferation and migration, increased sensitivity to apoptosis induced by interferon (IFN) or temozolomide in vitro , and inhibited GBM tumorigenesis in vivo . Here we show that ectopic expression of miR-203a in GBM cell lines promotes the IFN response pathway as evidenced by increased IFN production and IFN-stimulated gene (ISG) expression, and high basal tyrosine phosphorylation of multiple STAT proteins. Importantly, we identified that miR-203a directly suppressed the protein levels of ataxia-telangiectasia mutated (ATM) kinase that negatively regulates IFN production. We found that high ATM expression in GBM correlates with poor patient survival and that ATM expression is inversely correlated with miR-203a expression. Knockout of ATM expression and inhibition of ATM function in GBM cell lines inhibited cell proliferation and migration, increased sensitivity to apoptosis induced by therapeutic agents in vitro , and markedly suppressed GBM tumor growth and promoted animal survival. In contrast, restoring ATM levels in GBM cells ectopically expressing miR-203a increased tumorigenicity and decreased animal survival. Our study suggests that low miR-203a expression in GBM suppresses the interferon response through an ATM-dependent pathway.

Ectopic ERK Expression Induces Phenotypic Conversion of C10 Cells and Alters DNA Methyltransferase Expression

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sontag, Ryan L.; Weber, Thomas J.

2012-05-04

In some model systems constitutive extracellular signal regulated kinase (ERK) activation is sufficient to promote an oncogenic phenotype. Here we investigate whether constitutive ERK expression influences phenotypic conversion in murine C10 type II alveolar epithelial cells. C10 cells were stably transduced with an ERK1-green fluorescent protein (ERK1-GFP) chimera or empty vector and ectopic ERK expression was associated with the acquisition of soft agar focus-forming potential in late passage, but not early passage cells. Late passage ERK1-GFP cells exhibited a significant increase in the expression of DNA methyl transferases (DNMT1 and 3b) and a marked increase in sensitivity to 5-azacytidine (5-azaC)-mediatedmore » toxicity, relative to early passage ERK1-GFP cells and vector controls. The expression of xeroderma pigmentosum complementation group A (XPA) and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) were significantly increased in late passage cells, suggesting enhanced DNA damage recognition and repair activity which we interpret as a reflection of genomic instability. Phospho-ERK levels were dramatically decreased in late passage ERK1-GFP cells, relative to early passage and vector controls, and phospho-ERK levels were restored by treatment with sodium orthovanadate, indicating a role for phosphatase activity in this response. Collectively these observations suggest that ectopic ERK expression promotes phenotypic conversion of C10 cells that is associated with latent effects on epigenetic programming and phosphatase activities.« less

Effect of Plasmid Design and Type of Integration Event on Recombinant Protein Expression in Pichia pastoris.

PubMed

Vogl, Thomas; Gebbie, Leigh; Palfreyman, Robin W; Speight, Robert

2018-03-15

Pichia pastoris (syn. Komagataella phaffii ) is one of the most common eukaryotic expression systems for heterologous protein production. Expression cassettes are typically integrated in the genome to obtain stable expression strains. In contrast to Saccharomyces cerevisiae , where short overhangs are sufficient to target highly specific integration, long overhangs are more efficient in P. pastoris and ectopic integration of foreign DNA can occur. Here, we aimed to elucidate the influence of ectopic integration by high-throughput screening of >700 transformants and whole-genome sequencing of 27 transformants. Different vector designs and linearization approaches were used to mimic the most common integration events targeted in P. pastoris Fluorescence of an enhanced green fluorescent protein (eGFP) reporter protein was highly uniform among transformants when the expression cassettes were correctly integrated in the targeted locus. Surprisingly, most nonspecifically integrated transformants showed highly uniform expression that was comparable to specific integration, suggesting that nonspecific integration does not necessarily influence expression. However, a few clones (<10%) harboring ectopically integrated cassettes showed a greater variation spanning a 25-fold range, surpassing specifically integrated reference strains up to 6-fold. High-expression strains showed a correlation between increased gene copy numbers and high reporter protein fluorescence levels. Our results suggest that for comparing expression levels between strains, the integration locus can be neglected as long as a sufficient numbers of transformed strains are compared. For expression optimization of highly expressible proteins, increasing copy number appears to be the dominant positive influence rather than the integration locus, genomic rearrangements, deletions, or single-nucleotide polymorphisms (SNPs). IMPORTANCE Yeasts are commonly used as biotechnological production hosts for proteins and metabolites. In the yeast Saccharomyces cerevisiae , expression cassettes carrying foreign genes integrate highly specifically at the targeted sites in the genome. In contrast, cassettes often integrate at random genomic positions in nonconventional yeasts, such as Pichia pastoris (syn. Komagataella phaffii ). Hence, cells from the same transformation event often behave differently, with significant clonal variation necessitating the screening of large numbers of strains. The importance of this study is that we systematically investigated the influence of integration events in more than 700 strains. Our findings provide novel insight into clonal variation in P. pastoris and, thus, how to avoid pitfalls and obtain reliable results. The underlying mechanisms may also play a role in other yeasts and hence could be generally relevant for recombinant yeast protein production strains. Copyright © 2018 American Society for Microbiology.

The evolutionary duplication and probable demise of an endodermal GATA factor in Caenorhabditis elegans.

PubMed

Fukushige, Tetsunari; Goszczynski, Barbara; Tian, Helen; McGhee, James D

2003-10-01

We describe the elt-4 gene from the nematode Caenorhabditis elegans. elt-4 is predicted to encode a very small (72 residues, 8.1 kD) GATA-type zinc finger transcription factor. The elt-4 gene is located approximately 5 kb upstream of the C. elegans elt-2 gene, which also encodes a GATA-type transcription factor; the zinc finger DNA-binding domains are highly conserved (24/25 residues) between the two proteins. The elt-2 gene is expressed only in the intestine and is essential for normal intestinal development. This article explores whether elt-4 also has a role in intestinal development. Reporter fusions to the elt-4 promoter or reporter insertions into the elt-4 coding regions show that elt-4 is indeed expressed in the intestine, beginning at the 1.5-fold stage of embryogenesis and continuing into adulthood. elt-4 reporter fusions are also expressed in nine cells of the posterior pharynx. Ectopic expression of elt-4 cDNA within the embryo does not cause detectable ectopic expression of biochemical markers of gut differentiation; furthermore, ectopic elt-4 expression neither inhibits nor enhances the ectopic marker expression caused by ectopic elt-2 expression. A deletion allele of elt-4 was isolated but no obvious phenotype could be detected, either in the gut or elsewhere; brood sizes, hatching efficiencies, and growth rates were indistinguishable from wild type. We found no evidence that elt-4 provided backup functions for elt-2. We used microarray analysis to search for genes that might be differentially expressed between L1 larvae of the elt-4 deletion strain and wild-type worms. Paired hybridizations were repeated seven times, allowing us to conclude, with some confidence, that no candidate target transcript could be identified as significantly up- or downregulated by loss of elt-4 function. In vitro binding experiments could not detect specific binding of ELT-4 protein to candidate binding sites (double-stranded oligonucleotides containing single or multiple WGATAR sequences); ELT-4 protein neither enhanced nor inhibited the strong sequence-specific binding of the ELT-2 protein. Whereas ELT-2 protein is a strong transcriptional activator in yeast, ELT-4 protein has no such activity under similar conditions, nor does it influence the transcriptional activity of coexpressed ELT-2 protein. Although an elt-2 homolog was easily identified in the genomic sequence of the related nematode C. briggsae, no elt-4 homolog could be identified. Analysis of the changes in silent third codon positions within the DNA-binding domains indicates that elt-4 arose as a duplication of elt-2, some 25-55 MYA. Thus, elt-4 has survived far longer than the average duplicated gene in C. elegans, even though no obvious biological function could be detected. elt-4 provides an interesting example of a tandemly duplicated gene that may originally have been the same size as elt-2 but has gradually been whittled down to its present size of little more than a zinc finger. Although elt-4 must confer (or must have conferred) some selective advantage to C. elegans, we suggest that its ultimate evolutionary fate will be disappearance from the C. elegans genome.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Srisuttee, Ratakorn; Koh, Sang Seok; Department of Functional Genomics, University of Science and Technology, Daejeon 305-333

Highlights: Black-Right-Pointing-Pointer Up-regulation of SIRT1 protein and activity sensitizes Hep3B-HBX cells to oxidative stress-induced apoptosis. Black-Right-Pointing-Pointer Nuclear localization of SIRT1 is not required for oxidation-induced apoptosis. Black-Right-Pointing-Pointer Ectopic expression and enhanced activity of SIRT1 attenuate JNK phosphorylation. Black-Right-Pointing-Pointer Inhibition of SIRT1 activity restores resistance to oxidation-induced apoptosis through JNK activation. -- Abstract: We previously showed that SIRT1 deacetylase inhibits proliferation of hepatocellular carcinoma cells expressing hepatitis B virus (HBV) X protein (HBX), by destabilization of {beta}-catenin. Here, we report another role for SIRT1 in HBX-mediated resistance to oxidative stress. Ectopic expression and enhanced activity of SIRT1 sensitize Hep3B cells stablymore » expressing HBX to oxidative stress-induced apoptosis. SIRT1 mutant analysis showed that nuclear localization of SIRT1 is not required for sensitization of oxidation-mediated apoptosis. Furthermore, ectopic expression of SIRT1 and treatment with resveratrol (a SIRT1 activator) attenuated JNK phosphorylation, which is a prerequisite for resistance to oxidative stress-induced apoptosis. Conversely, suppression of SIRT1 activity with nicotinamide inhibited the effect of resveratrol on JNK phosphorylation, leading to restoration of resistance to oxidation-induced apoptosis. Taken together, these results suggest that up-regulation of SIRT1 under oxidative stress may be a therapeutic strategy for treatment of hepatocellular carcinoma cells related to HBV through inhibition of JNK activation.« less

Senseless, a Zn finger transcription factor, is necessary and sufficient for sensory organ development in Drosophila

NASA Technical Reports Server (NTRS)

Nolo, R.; Abbott, L. A.; Bellen, H. J.

2000-01-01

The senseless (sens) gene is required for proper development of most cell types of the embryonic and adult peripheral nervous system (PNS) of Drosophila. Sens is a nuclear protein with four Zn fingers that is expressed and required in the sensory organ precursors (SOP) for proper proneural gene expression. Ectopic expression of Sens in many ectodermal cells causes induction of PNS external sensory organ formation and is able to recreate an ectopic proneural field. Hence, sens is both necessary and sufficient for PNS development. Our data indicate that proneural genes activate sens expression. Sens is then in turn required to further activate and maintain proneural gene expression. This feedback mechanism is essential for selective enhancement and maintenance of proneural gene expression in the SOPs.

Tazarotene-Induced Gene 1 Interacts with DNAJC8 and Regulates Glycolysis in Cervical Cancer Cells.

PubMed

Wang, Chun-Hua; Shyu, Rong-Yaun; Wu, Chang-Chieh; Chen, Mao-Liang; Lee, Ming-Cheng; Lin, Yi-Yin; Wang, Lu-Kai; Jiang, Shun-Yuan; Tsai, Fu-Ming

2018-06-14

The tazarotene-induced gene 1 (TIG1) protein is a retinoidinducible growth regulator and is considered a tumor suppressor. Here, we show that DnaJ heat shock protein family member C8 (DNAJC8) is a TIG1 target that regulates glycolysis. Ectopic DNAJC8 expression induced the translocation of pyruvate kinase M2 (PKM2) into the nucleus, subsequently inducing glucose transporter 1 (GLUT1) expression to promote glucose uptake. Silencing either DNAJC8 or PKM2 alleviated the upregulation of GLUT1 expression and glucose uptake induced by ectopic DNAJC8 expression. TIG1 interacted with DNAJC8 in the cytosol, and this interaction completely blocked DNAJC8-mediated PKM2 translocation and inhibited glucose uptake. Furthermore, increased glycose uptake was observed in cells in which TIG1 was silenced. In conclusion, TIG1 acts as a pivotal repressor of DNAJC8 to enhance glucose uptake by partially regulating PKM2 translocation.

Ectopic expression of Msx2 in mammalian myotubes recapitulates aspects of amphibian muscle dedifferentiation.

PubMed

Yilmaz, Atilgan; Engeler, Rachel; Constantinescu, Simona; Kokkaliaris, Konstantinos D; Dimitrakopoulos, Christos; Schroeder, Timm; Beerenwinkel, Niko; Paro, Renato

2015-11-01

In contrast to urodele amphibians and teleost fish, mammals lack the regenerative responses to replace large body parts. Amphibian and fish regeneration uses dedifferentiation, i.e., reversal of differentiated state, as a means to produce progenitor cells to eventually replace damaged tissues. Therefore, induced activation of dedifferentiation responses in mammalian tissues holds an immense promise for regenerative medicine. Here we demonstrate that ectopic expression of Msx2 in cultured mouse myotubes recapitulates several aspects of amphibian muscle dedifferentiation. We found that MSX2, but not MSX1, leads to cellularization of myotubes and downregulates the expression of myotube markers, such as MHC, MRF4 and myogenin. RNA sequencing of myotubes ectopically expressing Msx2 showed downregulation of over 500 myotube-enriched transcripts and upregulation of over 300 myoblast-enriched transcripts. MSX2 selectively downregulated expression of Ptgs2 and Ptger4, two members of the prostaglandin pathway with important roles in myoblast fusion during muscle differentiation. Ectopic expression of Msx2, as well as Msx1, induced partial cell cycle re-entry of myotubes by upregulating CyclinD1 expression but failed to initiate S-phase. Finally, MSX2-induced dedifferentiation in mouse myotubes could be recapitulated by a pharmacological treatment with trichostatin A (TSA), bone morphogenetic protein 4 (BMP4) and fibroblast growth factor 1 (FGF1). Together, these observations indicate that MSX2 is a major driver of dedifferentiation in mammalian muscle cells. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

Proteomics Identification of Annexin A2 as a Key Mediator in the Metastasis and Proangiogenesis of Endometrial Cells in Human Adenomyosis*

PubMed Central

Zhou, Shengtao; Yi, Tao; Liu, Rui; Bian, Ce; Qi, Xiaorong; He, Xiang; Wang, Kui; Li, Jingyi; Zhao, Xia; Huang, Canhua; Wei, Yuquan

2012-01-01

Adenomyosis is a common estrogen-dependent disorder of females characterized by a downward extension of the endometrium into the uterine myometrium and neovascularization in ectopic lesions. It accounts for chronic pelvic pain, dysmenorrhea, menorrhagia, and infertility in 8.8–61.5% women worldwide. However, the molecular mechanisms for adenomyosis development remain poorly elucidated. Here, we utilized a two-dimensional polyacrylamide gel electrophoresis/MS-based proteomics analysis to compare and identify differentially expressed proteins in matched ectopic and eutopic endometrium of adenomyosis patients. A total of 93 significantly altered proteins were identified by tandem MS analysis. Further cluster analysis revealed a group of estrogen-responsive proteins as dysregulated in adenomyosis, among which annexin A2, a member of annexin family proteins, was found up-regulated most significantly in the ectopic endometrium of adenomyosis compared with its eutopic counterpart. Overexpression of ANXA2 was validated in ectopic lesions of human adenomyosis and was found to be tightly correlated with markers of epithelial to mesenchymal transition and dysmenorrhea severity of adenomyosis patients. Functional analysis demonstrated that estrogen could remarkably up-regulate ANXA2 and induce epithelial to mesenchymal transition in an in vitro adenomyosis model. Enforced expression of ANXA2 could mediate phenotypic mesenchymal-like cellular changes, with structural and functional alterations in a β-catenin/T-cell factor (Tcf) signaling-associated manner, which could be reversed by inhibition of ANXA2 expression. We also proved that enforced expression of ANXA2 enhanced the proangiogenic capacity of adenomyotic endometrial cells through HIF-1α/VEGF-A pathway. In vivo, we demonstrated that ANXA2 inhibition abrogated endometrial tissue growth, metastasis, and angiogenesis in an adenomyosis nude mice model and significantly alleviated hyperalgesia. Taken together, our data unraveled a dual role for ANXA2 in the pathogenesis of human adenomyosis through conferring endometrial cells both metastatic potential and proangiogenic capacity, which could serve as a potential therapeutic target for the treatment of adenomyosis patients. PMID:22493182

Vasostatin-2 inhibits cell proliferation and adhesion in vascular smooth muscle cells, which are associated with the progression of atherosclerosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hou, Jianghong, E-mail: jianghonghou@163.com; Xue, Xiaolin; Li, Junnong

2016-01-22

Recently, the serum expression level of vasostatin-2 was found to be reduced and is being studied as an important indicator to assess the presence and severity of coronary artery disease; the functional properties of vasostatin-2 and its relationship with the development of atherosclerosis remains unclear. In this study, we attempted to detect the expression of vasostatin-2 and its impact on human vascular smooth muscle cells (VSMCs). Quantitative real-time PCR (qRT-PCR) and western blot were used to assess the expression level of vasostatin-2 in VSMCs between those from atherosclerosis and disease-free donors; we found that vasostatin-2 was significantly down-regulated in atherosclerosismore » patient tissues and cell lines. In addition, the over-expression of vasostatin-2 apparently inhibits cell proliferation and migration in VSMCs. Gain-of-function in vitro experiments further show that vasostatin-2 over-expression significantly inhibits inflammatory cytokines release in VSMCs. In addition, cell adhesion experimental analysis showed that soluble adhesion molecules (sICAM-1, sVCAM-1) had decreased expression when vasostatin-2 was over-expressed in VSMCs. Therefore, our results indicate that vasostatin-2 is an atherosclerosis-related factor that can inhibit cell proliferation, inflammatory response and cell adhesion in VSMCs. Taken together, our results indicate that vasostatin-2 could serve as a potential diagnostic biomarker and therapeutic option for human atherosclerosis in the near future. - Highlights: • Vasostatin-2 levels were down-regulated in atherosclerosis patient tissues and VSMCs. • Ectopic expression of vasostatin-2 directly affects cell proliferation and migration in vitro. • Ectopic expression of vasostatin-2 protein affects pro-inflammatory cytokines release in VSMCs. • Ectopic expression of vasostatin-2 protein affects cell adhesion in VSMCs.« less

Interleukin-4 induces expression of eotaxin in endometriotic stromal cells.

PubMed

Ouyang, Zhuo; Osuga, Yutaka; Hirota, Yasushi; Hirata, Tetsuya; Yoshino, Osamu; Koga, Kaori; Yano, Tetsu; Taketani, Yuji

2010-06-01

To study the relationship between eotaxin and interleukin-4 (IL-4) in the pathophysiology of endometriosis. Comparative and laboratory study. University teaching hospital reproductive endocrinology and infertility practice. Ectopic endometrial tissues were collected from women with endometriosis. Ectopic endometrial stromal cells (ESCs) were isolated and cultured with IL-4. Ectopic endometriotic tissues were immunostained for eotaxin and IL-4. Gene expression of eotaxin was determined by standard and real-time reverse-transcriptase polymerase chain reaction. Secretion of eotaxin from ESC was measured using specific ELISA. The immunostained sections were examined. Interleukin-4 (IL-4) increased mRNA expression and protein secretion of eotaxin from ESC in a dose-dependent manner. Immunohistochemical analysis showed that eotaxin-positive cells colocalized with IL-4-positive cells and accumulated around the blood vessels in the stroma of endometriotic tissue. IL-4 induces eotaxin in ESCs, which might promote angiogenesis and the subsequent development of endometriosis. Copyright (c) 2010 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Cross-regulation in the mouse HoxB complex: the expression of Hoxb2 in rhombomere 4 is regulated by Hoxb1.

PubMed

Maconochie, M K; Nonchev, S; Studer, M; Chan, S K; Pöpperl, H; Sham, M H; Mann, R S; Krumlauf, R

1997-07-15

Correct regulation of the segment-restricted patterns of Hox gene expression is essential for proper patterning of the vertebrate hindbrain. We have examined the molecular basis of restricted expression of Hoxb2 in rhombomere 4 (r4), by using deletion analysis in transgenic mice to identify an r4 enhancer from the mouse gene. A bipartite Hox/Pbx binding motif is located within this enhancer, and in vitro DNA binding experiments showed that the vertebrate labial-related protein Hoxb1 will cooperatively bind to this site in a Pbx/Exd-dependent manner. The Hoxb2 r4 enhancer can be transactivated in vivo by the ectopic expression of Hoxb1, Hoxa1, and Drosophila labial in transgenic mice. In contrast, ectopic Hoxb2 and Hoxb4 are unable to induce expression, indicating that in vivo this enhancer preferentially responds to labial family members. Mutational analysis demonstrated that the bipartite Hox/Pbx motif is required for r4 enhancer activity and the responses to retinoids and ectopic Hox expression. Furthermore, three copies of the Hoxb2 motif are sufficient to mediate r4 expression in transgenic mouse embryos and a labial pattern in Drosophila embryos. This reporter expression in Drosophila embryos is dependent upon endogenous labial and exd, suggesting that the ability of this Hox/Pbx site to interact with labial-related proteins has been evolutionarily conserved. The endogenous Hoxb2 gene is no longer upregulated in r4 in Hoxb1 homozygous mutant embryos. On the basis of these experiments we conclude that the r4-restricted domain of Hoxb2 in the hindbrain is the result of a direct cross-regulatory interaction by Hoxb1 involving vertebrate Pbx proteins as cofactors. This suggests that part of the functional role of Hoxb1 in maintaining r4 identity may be mediated by the Hoxb2 gene.

mRNA-binding protein TIA-1 reduces cytokine expression in human endometrial stromal cells and is down-regulated in ectopic endometrium.

PubMed

Karalok, Hakan Mete; Aydin, Ebru; Saglam, Ozlen; Torun, Aysenur; Guzeloglu-Kayisli, Ozlem; Lalioti, Maria D; Kristiansson, Helena; Duke, Cindy M P; Choe, Gina; Flannery, Clare; Kallen, Caleb B; Seli, Emre

2014-12-01

Cytokines and growth factors play important roles in endometrial function and the pathogenesis of endometriosis. mRNAs encoding cytokines and growth factors undergo rapid turnover; primarily mediated by adenosine- and uridine-rich elements (AREs) located in their 3'-untranslated regions. T-cell intracellular antigen (TIA-1), an mRNA-binding protein, binds to AREs in target transcripts, leading to decreased gene expression. The purpose of this article was to determine whether TIA-1 plays a role in the regulation of endometrial cytokine and growth factor expression during the normal menstrual cycle and whether TIA-1 expression is altered in women with endometriosis. Eutopic endometrial tissue obtained from women without endometriosis (n = 30) and eutopic and ectopic endometrial tissues from women with endometriosis (n = 17) were immunostained for TIA-1. Staining intensities were evaluated by histological scores (HSCOREs). The regulation of endometrial TIA-1 expression by immune factors and steroid hormones was studied by treating primary cultured human endometrial stromal cells (HESCs) with vehicle, lipopolysaccharide, TNF-α, IL-6, estradiol, or progesterone, followed by protein blot analyses. HESCs were engineered to over- or underexpress TIA-1 to test whether TIA-1 regulates IL-6 or TNF-α expression in these cells. We found that TIA-1 is expressed in endometrial stromal and glandular cells throughout the menstrual cycle and that this expression is significantly higher in the perimenstrual phase. In women with endometriosis, TIA-1 expression in eutopic and ectopic endometrium was reduced compared with TIA-1 expression in eutopic endometrium of unaffected control women. Lipopolysaccharide and TNF-α increased TIA-1 expression in HESCs in vitro, whereas IL-6 or steroid hormones had no effect. In HESCs, down-regulation of TIA-1 resulted in elevated IL-6 and TNF-α expression, whereas TIA-1 overexpression resulted in decreased IL-6 and TNF-α expression. Endometrial TIA-1 is regulated throughout the menstrual cycle, TIA-1 modulates the expression of immune factors in endometrial cells, and downregulation of TIA-1 may contribute to the pathogenesis of endometriosis.

mRNA-Binding Protein TIA-1 Reduces Cytokine Expression in Human Endometrial Stromal Cells and Is Down-Regulated in Ectopic Endometrium

PubMed Central

Karalok, Hakan Mete; Aydin, Ebru; Saglam, Ozlen; Torun, Aysenur; Guzeloglu-Kayisli, Ozlem; Lalioti, Maria D.; Kristiansson, Helena; Duke, Cindy M. P.; Choe, Gina; Flannery, Clare; Kallen, Caleb B.

2014-01-01

Background: Cytokines and growth factors play important roles in endometrial function and the pathogenesis of endometriosis. mRNAs encoding cytokines and growth factors undergo rapid turnover; primarily mediated by adenosine- and uridine-rich elements (AREs) located in their 3′-untranslated regions. T-cell intracellular antigen (TIA-1), an mRNA-binding protein, binds to AREs in target transcripts, leading to decreased gene expression. Objective: The purpose of this article was to determine whether TIA-1 plays a role in the regulation of endometrial cytokine and growth factor expression during the normal menstrual cycle and whether TIA-1 expression is altered in women with endometriosis. Methods: Eutopic endometrial tissue obtained from women without endometriosis (n = 30) and eutopic and ectopic endometrial tissues from women with endometriosis (n = 17) were immunostained for TIA-1. Staining intensities were evaluated by histological scores (HSCOREs). The regulation of endometrial TIA-1 expression by immune factors and steroid hormones was studied by treating primary cultured human endometrial stromal cells (HESCs) with vehicle, lipopolysaccharide, TNF-α, IL-6, estradiol, or progesterone, followed by protein blot analyses. HESCs were engineered to over- or underexpress TIA-1 to test whether TIA-1 regulates IL-6 or TNF-α expression in these cells. Results: We found that TIA-1 is expressed in endometrial stromal and glandular cells throughout the menstrual cycle and that this expression is significantly higher in the perimenstrual phase. In women with endometriosis, TIA-1 expression in eutopic and ectopic endometrium was reduced compared with TIA-1 expression in eutopic endometrium of unaffected control women. Lipopolysaccharide and TNF-α increased TIA-1 expression in HESCs in vitro, whereas IL-6 or steroid hormones had no effect. In HESCs, down-regulation of TIA-1 resulted in elevated IL-6 and TNF-α expression, whereas TIA-1 overexpression resulted in decreased IL-6 and TNF-α expression. Conclusions: Endometrial TIA-1 is regulated throughout the menstrual cycle, TIA-1 modulates the expression of immune factors in endometrial cells, and downregulation of TIA-1 may contribute to the pathogenesis of endometriosis. PMID:25140393

Ectopic expression of the gastric inhibitory polypeptide receptor gene is a sufficient genetic event to induce benign adrenocortical tumor in a xenotransplantation model.

PubMed

Mazzuco, Tania L; Chabre, Olivier; Sturm, Nathalie; Feige, Jean-Jacques; Thomas, Michaël

2006-02-01

Aberrant expression of ectopic G protein-coupled receptors (GPCRs) in adrenal cortex tissue has been observed in several cases of ACTH-independent macronodular adrenal hyperplasias and adenomas associated with Cushing's syndrome. Although there is clear clinical evidence for the implication of these ectopic receptors in abnormal regulation of cortisol production, whether this aberrant GPCR expression is the cause or the consequence of the development of an adrenal hyperplasia is still an open question. To answer it, we genetically engineered primary bovine adrenocortical cells to have them express the gastric inhibitory polypeptide receptor. After transplantation of these modified cells under the renal capsule of adrenalectomized immunodeficient mice, tissues formed had their functional and histological characteristics analyzed. We observed the formation of an enlarged and hyperproliferative adenomatous adrenocortical tissue that secreted cortisol in a gastric inhibitory polypeptide-dependent manner and induced a mild Cushing's syndrome with hyperglycemia. Moreover, we show that tumor development was ACTH independent. Thus, a single genetic event, inappropriate expression of a nonmutated GPCR gene, is sufficient to initiate the complete phenotypic alterations that ultimately lead to the formation of a benign adrenocortical tumor.

Ectopical expression of FABP4 gene can induce bovine muscle-derived stem cells adipogenesis.

PubMed

Zhang, Le; Zhao, Yanfang; Ning, Yue; Wang, Hongbao; Zan, Linsen

2017-01-08

Fatty acid binding protein 4 (FABP4) plays a key role in Fatty acid catabolism in mammals. Findings from our previous studies have indicated that FABP4 neither affect the differentiation of bovine preadipocytes nor does it change the expression of upstream genes. To investigate whether ectopically expressed FABP4 can induces Muscle-Derived Stem Cells (MDSCs) lipid synthesis and understand the regulatory mechanism behind it. In this study, adenoviruses infection is achieved to ectopically expressed FABP4 in bovine MDSCs, RNA-seq analyses at the very early stages of induction were performed to reveal gene expression level changes during MDSCs transdifferentiation. Results showed FABP4 can induce bovine Muscle-Derived Stem Cells transdifferentiation into adipocyte-like cells, 23 genes' expression levels changed after 24 h inducing although there is no significant change in cell phenotypes. Along with induction time, more differently expressed genes (256 genes changes after 48 h induction) were screened out. These genes should be at the downstream of signal pathways and be regulated by the 23 genes identified before. Our findings may provide a unique new model for studying the molecular control of cattle cross-talk between adipose and skeletal muscle. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Signaling by ectopically expressed Drosophila Src64 requires the protein-tyrosine phosphatase corkscrew and the adapter downstream of receptor kinases.

PubMed

Cooper, J A; Simon, M A; Kussick, S J

1996-11-01

Vertebrate Src can be activated by specific mutations to become oncogenic. Analogous mutations in Drosophila Src64 (DSrc) induce abnormal differentiation of photoreceptor cells when expressed ectopically in the developing Drosophila adult eye. We have investigated the roles that the adapter protein, Downstream of receptor kinases (Drk), and the SH2 domain-containing tyrosine phosphatase, Corkscrew (Csw), play in this process. We find that dominant-negative mutations in either the drk or csw genes ameliorate the developmental abnormalities induced by activated DSrc. This suggests that Drk and Csw are required downstream of, or parallel to, DSrc. Csw does not act solely as an upstream activator of DSrc. The results are discussed in relation to potential roles for the vertebrate homologues of Drk and Csw (Grb2 and SHP2, respectively) in the transformation of fibroblasts by vertebrate Src.

The homeodomain transcription factor Cdx1 does not behave as an oncogene in normal mouse intestine.

PubMed

Crissey, Mary Ann S; Guo, Rong-Jun; Fogt, Franz; Li, Hong; Katz, Jonathan P; Silberg, Debra G; Suh, Eun Ran; Lynch, John P

2008-01-01

The Caudal-related homeobox genes Cdx1 and Cdx2 are intestine-specific transcription factors that regulate differentiation of intestinal cell types. Previously, we have shown Cdx1 to be antiproliferative and to promote cell differentiation. However, other studies have suggested that Cdx1 may be an oncogene. To test for oncogenic behavior, we used the murine villin promoter to ectopically express Cdx1 in the small intestinal villi and colonic surface epithelium. No changes in intestinal architecture, cell differentiation, or lineage selection were observed with expression of the transgene. Classic oncogenes enhance proliferation and induce tumors when ectopically expressed. However, the Cdx1 transgene neither altered intestinal proliferation nor induced spontaneous intestinal tumors. In a murine model for colitis-associated cancer, the Cdx1 transgene decreased, rather than increased, the number of adenomas that developed. In the polyps, the expression of the endogenous and the transgenic Cdx1 proteins was largely absent, whereas endogenous Villin expression was retained. This suggests that transgene silencing was specific and not due to a general Villin inactivation. In conclusion, neither the ectopic expression of Cdx1 was associated with changes in intestinal cell proliferation or differentiation nor was there increased intestinal cancer susceptibility. Our results therefore suggest that Cdx1 is not an oncogene in normal intestinal epithelium.

Translational repression by an RNA-binding protein promotes differentiation to infective forms in Trypanosoma cruzi.

PubMed

Romaniuk, Maria Albertina; Frasch, Alberto Carlos; Cassola, Alejandro

2018-06-01

Trypanosomes, protozoan parasites of medical importance, essentially rely on post-transcriptional mechanisms to regulate gene expression in insect vectors and vertebrate hosts. RNA binding proteins (RBPs) that associate to the 3'-UTR of mature mRNAs are thought to orchestrate master developmental programs for these processes to happen. Yet, the molecular mechanisms by which differentiation occurs remain largely unexplored in these human pathogens. Here, we show that ectopic inducible expression of the RBP TcUBP1 promotes the beginning of the differentiation process from non-infective epimastigotes to infective metacyclic trypomastigotes in Trypanosoma cruzi. In early-log epimastigotes TcUBP1 promoted a drop-like phenotype, which is characterized by the presence of metacyclogenesis hallmarks, namely repositioning of the kinetoplast, the expression of an infective-stage virulence factor such as trans-sialidase, increased resistance to lysis by human complement and growth arrest. Furthermore, TcUBP1-ectopic expression in non-infective late-log epimastigotes promoted full development into metacyclic trypomastigotes. TcUBP1-derived metacyclic trypomastigotes were infective in cultured cells, and developed normally into amastigotes in the cytoplasm. By artificial in vivo tethering of TcUBP1 to the 3' untranslated region of a reporter mRNA we were able to determine that translation of the reporter was reduced by 8-fold, while its mRNA abundance was not significantly compromised. Inducible ectopic expression of TcUBP1 confirmed its role as a translational repressor, revealing significant reduction in the translation rate of multiple proteins, a reduction of polysomes, and promoting the formation of mRNA granules. Expression of TcUBP1 truncated forms revealed the requirement of both N and C-terminal glutamine-rich low complexity sequences for the development of the drop-like phenotype in early-log epimastigotes. We propose that a rise in TcUBP1 levels, in synchrony with nutritional deficiency, can promote the differentiation of T. cruzi epimastigotes into infective metacyclic trypomastigotes.

The myogenic repressor gene Holes in muscles is a direct transcriptional target of Twist and Tinman in the Drosophila embryonic mesoderm

PubMed Central

Elwell, Jennifer A.; Lovato, TyAnna L.; Adams, Melanie M.; Baca, Erica M.; Lee, Thai; Cripps, Richard M.

2015-01-01

Understanding the regulatory circuitry controlling myogenesis is critical to understanding developmental mechanisms and developmentally-derived diseases. We analyzed the transcriptional regulation of a Drosophila myogenic repressor gene, Holes in muscles (Him). Previously, Him was shown to inhibit Myocyte enhancer factor-2 (MEF2) activity, and is expressed in myoblasts but not differentiating myotubes. We demonstrate that different phases of Him embryonic expression arise through the actions of different enhancers, and we characterize the enhancer required for its early mesoderm expression. This Him early mesoderm enhancer contains two conserved binding sites for the basic helix-loop-helix regulator Twist, and one binding site for the NK homeodomain protein Tinman. The sites for both proteins are required for enhancer activity in early embryos. Twist and Tinman activate the enhancer in tissue culture assays, and ectopic expression of either factor is sufficient to direct ectopic expression of a Him-lacZ reporter, or of the endogenous Him gene. Moreover, sustained expression of twist expression in the mesoderm up-regulates mesodermal Him expression in late embryos. Our findings provide a model to define mechanistically how Twist can both promotes myogenesis through direct activation of Mef2, and can place a brake on myogenesis, through direct activation of Him. PMID:25704510

Elevated ATF4 Expression, in the Absence of Other Signals, Is Sufficient for Transcriptional Induction via CCAAT Enhancer-binding Protein-activating Transcription Factor Response Elements*

PubMed Central

Shan, Jixiu; Örd, Daima; Örd, Tõnis; Kilberg, Michael S.

2009-01-01

Protein limitation in vivo or amino acid deprivation of cells in culture causes a signal transduction cascade consisting of activation of the kinase GCN2 (general control nonderepressible 2), phosphorylation of eukaryotic initiation factor 2, and increased synthesis of activating transcription factor (ATF) 4 by a translational control mechanism. In a self-limiting transcriptional program, ATF4 transiently activates a wide range of downstream target genes involved in transport, cellular metabolism, and other cell functions. Simultaneous activation of other signal transduction pathways by amino acid deprivation led to the question of whether or not the increased abundance of ATF4 alone was sufficient to trigger the transcriptional control mechanisms. Using 293 cells that ectopically express ATF4 in a tetracycline-inducible manner showed that ATF4 target genes were activated in the absence of amino acid deprivation. Ectopic expression of ATF4 alone resulted in effective recruitment of the general transcription machinery, but some reduction in histone modification was observed. These data document that ATF4 alone is sufficient to trigger the amino acid-responsive transcriptional control program. However, the absolute amount of ectopic ATF4 required to achieve the same degree of transcriptional activation observed after amino acid limitation was greater, suggesting that other factors may serve to enhance ATF4 function. PMID:19509279

Drosophila bunched integrates opposing DPP and EGF signals to set the operculum boundary.

PubMed

Dobens, L L; Peterson, J S; Treisman, J; Raftery, L A

2000-02-01

The Drosophila BMP homolog DPP can function as a morphogen, inducing multiple cell fates across a developmental field. However, it is unknown how graded levels of extracellular DPP are interpreted to organize a sharp boundary between different fates. Here we show that opposing DPP and EGF signals set the boundary for an ovarian follicle cell fate. First, DPP regulates gene expression in the follicle cells that will create the operculum of the eggshell. DPP induces expression of the enhancer trap reporter A359 and represses expression of bunched, which encodes a protein similar to the mammalian transcription factor TSC-22. Second, DPP signaling indirectly regulates A359 expression in these cells by downregulating expression of bunched. Reduced bunched function restores A359 expression in cells that lack the Smad protein MAD; ectopic expression of BUNCHED suppresses A359 expression in this region. Importantly, reduction of bunched function leads to an expansion of the operculum and loss of the collar at its boundary. Third, EGF signaling upregulates expression of bunched. We previously demonstrated that the bunched expression pattern requires the EGF receptor ligand GURKEN. Here we show that activated EGF receptor is sufficient to induce ectopic bunched expression. Thus, the balance of DPP and EGF signals sets the boundary of bunched expression. We propose that the juxtaposition of cells with high and low BUNCHED activity organizes a sharp boundary for the operculum fate.

Differential gene expression in Ndph-knockout mice in retinal development.

PubMed

Schäfer, Nikolaus F; Luhmann, Ulrich F O; Feil, Silke; Berger, Wolfgang

2009-02-01

Mutations in the NDP gene impair angiogenesis in the eyes of patients diagnosed with a type of blindness belonging to the group of exudative vitreoretinopathies. This study was conducted to investigate the differential gene expression caused by the absence of Norrin (the NDP protein) in the developing mouse retina and to elucidate early pathogenic events. A comparative gene expression analysis was performed on postnatal day (p)7 retinas from a knockout mouse model for Norrie disease using gene microarrays. Subsequently, results were verified by quantitative real-time PCR analyses. Immunohistochemistry was performed for the vascular permeability marker plasmalemma vesicle associated protein (Plvap). Our study identified expression differences in Ndph(y/-) versus wild-type mice retinas at p7. Gene transcription of the neutral amino acid transporter Slc38a5, apolipoprotein D (ApoD), and angiotensin II receptor-like 1 (Agtrl1) was decreased in the knockout mouse, whereas transcript levels of adrenomedullin (Adm) and of the plasmalemma vesicle associated protein (Plvap) were increased in comparison to the wild-type. In addition, ectopic expression of Plvap was found in the developing retinal vasculature of Norrin-knockout mice on the protein level. These data provide molecular evidence for a role of Norrin in the development of the retinal vasculature. Expression of two genes, Plvap and Slc38a5, is considerably altered in retinal development of Norrin-knockout mice and may reflect or contribute to the pathogenesis of the disease. In particular, ectopic expression of Plvap is consistent with hallmark disease symptoms in mice and humans.

Induced stem cell neoplasia in a cnidarian by ectopic expression of a POU domain transcription factor.

PubMed

Millane, R Cathriona; Kanska, Justyna; Duffy, David J; Seoighe, Cathal; Cunningham, Stephen; Plickert, Günter; Frank, Uri

2011-06-01

The evolutionary origin of stem cell pluripotency is an unresolved question. In mammals, pluripotency is limited to early embryos and is induced and maintained by a small number of key transcription factors, of which the POU domain protein Oct4 is considered central. Clonal invertebrates, by contrast, possess pluripotent stem cells throughout their life, but the molecular mechanisms that control their pluripotency are poorly defined. To address this problem, we analyzed the expression pattern and function of Polynem (Pln), a POU domain gene from the marine cnidarian Hydractinia echinata. We show that Pln is expressed in the embryo and adult stem cells of the animal and that ectopic expression in epithelial cells induces stem cell neoplasms and loss of epithelial tissue. Neoplasm cells downregulated the transgene but expressed the endogenous Pln gene and also Nanos, Vasa, Piwi and Myc, which are all known cnidarian stem cell markers. Retinoic acid treatment caused downregulation of Pln and the differentiation of neoplasm cells to neurosensory and epithelial cells. Pln downregulation by RNAi led to differentiation. Collectively, our results suggest an ancient role of POU proteins as key regulators of animal stem cells.

MiR-17/20/93/106 promote hematopoietic cell expansion by targeting sequestosome 1–regulated pathways in mice

PubMed Central

Meenhuis, Annemarie; van Veelen, Peter A.; de Looper, Hans; van Boxtel, Nicole; van den Berge, Iris J.; Sun, Su M.; Taskesen, Erdogan; Stern, Patrick; de Ru, Arnoud H.; van Adrichem, Arjan J.; Demmers, Jeroen; Jongen-Lavrencic, Mojca; Löwenberg, Bob; Touw, Ivo P.; Sharp, Phillip A.

2011-01-01

MicroRNAs (miRNAs) are pivotal for regulation of hematopoiesis but their critical targets remain largely unknown. Here, we show that ectopic expression of miR-17, -20,-93 and -106, all AAAGUGC seed-containing miRNAs, increases proliferation, colony outgrowth and replating capacity of myeloid progenitors and results in enhanced P-ERK levels. We found that these miRNAs are endogenously and abundantly expressed in myeloid progenitors and down-regulated in mature neutrophils. Quantitative proteomics identified sequestosome 1 (SQSTM1), an ubiquitin-binding protein and regulator of autophagy-mediated protein degradation, as a major target for these miRNAs in myeloid progenitors. In addition, we found increased expression of Sqstm1 transcripts during CSF3-induced neutrophil differentiation of 32D-CSF3R cells and an inverse correlation of SQSTM1 protein levels and miR-106 expression in AML samples. ShRNA-mediated silencing of Sqstm1 phenocopied the effects of ectopic miR-17/20/93/106 expression in hematopoietic progenitors in vitro and in mice. Further, SQSTM1 binds to the ligand-activated colony-stimulating factor 3 receptor (CSF3R) mainly in the late endosomal compartment, but not in LC3 positive autophagosomes. SQSTM1 regulates CSF3R stability and ligand-induced mitogen-activated protein kinase signaling. We demonstrate that AAAGUGC seed-containing miRNAs promote cell expansion, replating capacity and signaling in hematopoietic cells by interference with SQSTM1-regulated pathways. PMID:21628417

Analysis of the oncogene BRAF mutation and the correlation of the expression of wild-type BRAF and CREB1 in endometriosis

PubMed Central

Lv, Xiao; Ma, Yue; Long, Zaiqiu

2018-01-01

B-Raf proto-oncogene, serine/threonine kinase (BRAF) has previously been identified as a candidate target gene in endometriosis. Wild-type and mutated BRAF serve important roles in different diseases. The aim of the present study was to explore BRAF mutation, the mRNA and protein expression of wild-type BRAF (wtBRAF) in endometriosis, and the association between the expression levels of wtBRAF and the predicted transcription factor cAMP responsive element binding protein 1 (CREB1). In the present study, BRAF mutation was detected using Sanger sequencing among 30 ectopic and matched eutopic endometrium samples of patients with endometriosis as well as 25 normal endometrium samples, and no BRAF mutation was detected in exons 11 or 15. A region of ~2,000 bp upstream of the BRAF gene was then screened using NCBI and UCSC databases, and CREB1 was identified as a potential transcription factor of BRAF by analysis with the JASPAR and the TRANSFAC databases. Quantitative polymerase chain reaction was used to analysis the mRNA expression levels of wtBRAF and CREB1, and the corresponding protein expression levels were evaluated using immunohistochemistry and western blot analysis. The results revealed that the mRNA and protein expression levels of wtBRAF and CREB1 were significantly upregulated in the eutopic endometrial tissues of patients with endometriosis compared with normal endometrial tissues (P<0.05) and no significant difference in wtBRAF and CREB1 levels was detected between the ectopic and eutopic endometrium (P>0.05). In addition, correlation analysis revealed that the protein expression of CREB1 was positively correlated with the transcript level and protein expression of wtBRAF. It is reasonable to speculate that CREB1 may activate the transcription of wtBRAF through directly binding to its promoter, increasing BRAF expression and regulating the cell proliferation, migration and invasion of endometriosis. PMID:29286077

Brca1 regulates in vitro differentiation of mammary epithelial cells.

PubMed

Kubista, Marion; Rosner, Margit; Kubista, Ernst; Bernaschek, Gerhard; Hengstschläger, Markus

2002-07-18

Murine Brca1 is widely expressed during development in different tissues. Why alterations of BRCA1 lead specifically to breast and ovarian cancer is currently not clarified. Here we show that Brca1 protein expression is upregulated during mammary epithelial differentiation of HC11 cells, during differentiation of C2C12 myoblasts into myotubes and during neuronal differentiation of N1E-115 cells. Ectopic overexpression of BRCA1 and downregulation of endogenous Brca1 expression specifically affect the regulation of mammary epithelial cell differentiation. Accelerated mammary epithelial cell differentiation upon high ectopic BRCA1 expression is not a consequence of the anti-proliferative capacity of this tumor suppressor and independent of functional p53. Overexpression of the BRCA1 variant lacking the large central exon 11 has no effects on mammary epithelial cell differentiation. These data provide new insights into the cellular role of Brca1.

Combined VEGF and LMP-1 delivery enhances osteoprogenitor cell differentiation and ectopic bone formation.

PubMed

Wang, Xiuli; Cui, Fuai; Madhu, Vedavathi; Dighe, Abhijit S; Balian, Gary; Cui, Quanjun

2011-02-01

A novel strategy to enhance bone repair is to combine angiogenic factors and osteogenic factors. We combined vascular endothelial growth factor (VEGF) and LIM mineralization protein-1 (LMP-1) by using an internal ribosome entry site to link the genes within a single plasmid. We then evaluated the effects on osteoblastic differentiation in vitro and ectopic bone formation in vivo with a subcutaneously placed PLAGA scaffold loaded with a cloned mouse osteoprogenitor cell line, D1, transfected with plasmids containing VEGF and LMP-1 genes. The cells expressing both genes elevated mRNA expression of RunX2 and β-catenin and alkaline phosphatase activity compared to cells from other groups. In vivo, X-ray and micro-CT analysis of the retrieved implants revealed more ectopic bone formation at 2 and 3 weeks but not at 4 weeks compared to other groups. The results indicate that the combination of the therapeutic growth factors potentiates cell differentiation and may promote osteogenesis.

Planar cell polarity controls directional Notch signaling in the Drosophila leg

PubMed Central

Capilla, Amalia; Johnson, Ruth; Daniels, Maki; Benavente, María; Bray, Sarah J.; Galindo, Máximo Ibo

2012-01-01

The generation of functional structures during development requires tight spatial regulation of signaling pathways. Thus, in Drosophila legs, in which Notch pathway activity is required to specify joints, only cells distal to ligand-producing cells are capable of responding. Here, we show that the asymmetric distribution of planar cell polarity (PCP) proteins correlates with this spatial restriction of Notch activation. Frizzled and Dishevelled are enriched at distal sides of each cell and hence localize at the interface with ligand-expressing cells in the non-responding cells. Elimination of PCP gene function in cells proximal to ligand-expressing cells is sufficient to alleviate the repression, resulting in ectopic Notch activity and ectopic joint formation. Mutations that compromise a direct interaction between Dishevelled and Notch reduce the efficacy of repression. Likewise, increased Rab5 levels or dominant-negative Deltex can suppress the ectopic joints. Together, these results suggest that PCP coordinates the spatial activity of the Notch pathway by regulating endocytic trafficking of the receptor. PMID:22736244

Ectopic transgene expression in the retina of four transgenic mouse lines

PubMed Central

Gábriel, Robert; Erdélyi, Ferenc; Szabó, Gábor; Lawrence, J. Josh

2017-01-01

Retinal expression of transgenes was examined in four mouse lines. Two constructs were driven by the choline acetyltransferase (ChAT) promoter: green fluorescent protein conjugated to tau protein (tau-GFP) or cytosolic yellow fluorescent protein (YFP) generated through CRE recombinase-induced expression of Rosa26 (ChAT-CRE/ Rosa26YFP). Two other constructs targeted inhibitory interneurons: GABAergic horizontal and amacrine cells identified by glutamic acid decarboxylase (GAD65-GFP) or parvalbumin (PV) cells (PV-CRE/Rosa26YFP). Animals were transcardially perfused and retinal sections prepared. Antibodies against PV, calretinin (CALR), calbindin (CALB), and tyrosine hydroxylase (TH) were used to counterstain transgene-expressing cells. In PVxRosa and ChAT-tauGFP constructs, staining appeared in vertically oriented row of processes resembling Müller cells. In the ChATxRosa construct, populations of amacrine cells and neurons in the ganglion cell layer were labeled. Some cones also exhibited GFP fluorescence. CALR, PV and TH were found in none of these cells. Occasionally, we found GFP/ CALR and GFP/PV double-stained cells in the ganglion cell layer (GCL). In the GAD65-GFP construct, all layers of the neuroretina were labeled, except photoreceptors. Not all horizontal cells expressed GFP. We did not find GFP/TH double-labeled cells and GFP was rarely present in CALR-and CALB-containing cells. Many PV-positive neurons were also labeled for GFP, including small diameter amacrines. In the GCL, single labeling for GFP and PV was ascertained, as well as several CALR/PV double-stained neurons. In the GCL, cells triple labeled with GFP/CALR/ CALB were sparse. In conclusion, only one of the four transgenic constructs exhibited an expression pattern consistent with endogenous retinal protein expression, while the others strongly suggested ectopic gene expression. PMID:26563404

Ectopic expression of Arabidopsis Target of Rapamycin (AtTOR) improves water-use efficiency and yield potential in rice

NASA Astrophysics Data System (ADS)

Bakshi, Achala; Moin, Mazahar; Kumar, M. Udaya; Reddy, Aramati Bindu Madhava; Ren, Maozhi; Datla, Raju; Siddiq, E. A.; Kirti, P. B.

2017-02-01

The target of Rapamycin (TOR) present in all eukaryotes is a multifunctional protein, regulating growth, development, protein translation, ribosome biogenesis, nutrient, and energy signaling. In the present study, ectopic expression of TOR gene of Arabidopsis thaliana in a widely cultivated indica rice resulted in enhanced plant growth under water-limiting conditions conferring agronomically important water-use efficiency (WUE) trait. The AtTOR high expression lines of rice exhibited profuse tillering, increased panicle length, increased plant height, high photosynthetic efficiency, chlorophyll content and low Δ13C. Δ13C, which is inversely related to high WUE, was as low as 17‰ in two AtTOR high expression lines. These lines were also insensitive to the ABA-mediated inhibition of seed germination. The significant upregulation of 15 stress-specific genes in high expression lines indicates their contribution to abiotic stress tolerance. The constitutive expression of AtTOR is also associated with significant transcriptional upregulation of putative TOR complex-1 components, OsRaptor and OsLST8. Glucose-mediated transcriptional activation of AtTOR gene enhanced lateral root formation. Taken together, our findings indicate that TOR, in addition to its multiple cellular functions, also plays an important role in response to abiotic stress and potentially enhances WUE and yield related attributes.

Growth inhibition mediated by PSP94 or CRISP-3 is prostate cancer cell line specific.

PubMed

Pathak, Bhakti R; Breed, Ananya A; Nakhawa, Vaishali H; Jagtap, Dhanashree D; Mahale, Smita D

2010-09-01

The prostate secretory protein of 94 amino acids (PSP94) has been shown to interact with cysteine-rich secretory protein 3 (CRISP-3) in human seminal plasma. Interestingly, PSP94 expression is reduced or lost in the majority of the prostate tumours, whereas CRISP-3 expression is upregulated in prostate cancer compared with normal prostate tissue. To obtain a better understanding of the individual roles these proteins have in prostate tumourigenesis and the functional relevance of their interaction, we ectopically expressed either PSP94 or CRISP-3 alone or PSP94 along with CRISP-3 in three prostate cell lines (PC3, WPE1-NB26 and LNCaP) and performed growth inhibition assays. Reverse transcription-polymerase chain reaction and Western blot analysis were used to screen prostate cell lines for PSP94 and CRISP-3 expression. Mammalian expression constructs for human PSP94 and CRISP-3 were also generated and the expression, localization and secretion of recombinant protein were assayed by transfection followed by Western blot analysis and immunofluorescence assay. The effect that ectopic expression of PSP94 or CRISP-3 had on cell growth was studied by clonogenic survival assay following transfection. To evaluate the effects of co-expression of the two proteins, stable clones of PC3 that expressed PSP94 were generated. They were subsequently transfected with a CRISP-3 expression construct and subjected to clonogenic survival assay. Our results showed that PSP94 and CRISP-3 could each induce growth inhibition in a cell line specific manner. Although the growth of CRISP-3-positive cell lines was inhibited by PSP94, growth inhibition mediated by CRISP-3 was not affected by the presence or absence of PSP94. This suggests that CRISP-3 may participate in PSP94-independent activities during prostate tumourigenesis.

Targeting MYCN-Driven Transcription By BET-Bromodomain Inhibition.

PubMed

Henssen, Anton; Althoff, Kristina; Odersky, Andrea; Beckers, Anneleen; Koche, Richard; Speleman, Frank; Schäfers, Simon; Bell, Emma; Nortmeyer, Maike; Westermann, Frank; De Preter, Katleen; Florin, Alexandra; Heukamp, Lukas; Spruessel, Annika; Astrahanseff, Kathy; Lindner, Sven; Sadowski, Natalie; Schramm, Alexander; Astorgues-Xerri, Lucile; Riveiro, Maria E; Eggert, Angelika; Cvitkovic, Esteban; Schulte, Johannes H

2016-05-15

Targeting BET proteins was previously shown to have specific antitumoral efficacy against MYCN-amplified neuroblastoma. We here assess the therapeutic efficacy of the BET inhibitor, OTX015, in preclinical neuroblastoma models and extend the knowledge on the role of BRD4 in MYCN-driven neuroblastoma. The efficacy of OTX015 was assessed in in vitro and in vivo models of human and murine MYCN-driven neuroblastoma. To study the effects of BET inhibition in the context of high MYCN levels, MYCN was ectopically expressed in human and murine cells. The effect of OTX015 on BRD4-regulated transcriptional pause release was analyzed using BRD4 and H3K27Ac chromatin immunoprecipitation coupled with DNA sequencing (ChIP-Seq) and gene expression analysis in neuroblastoma cells treated with OTX015 compared with vehicle control. OTX015 showed therapeutic efficacy against preclinical MYCN-driven neuroblastoma models. Similar to previously described BET inhibitors, concurrent MYCN repression was observed in OTX015-treated samples. Ectopic MYCN expression, however, did not abrogate effects of OTX015, indicating that MYCN repression is not the only target of BET proteins in neuroblastoma. When MYCN was ectopically expressed, BET inhibition still disrupted MYCN target gene transcription without affecting MYCN expression. We found that BRD4 binds to super-enhancers and MYCN target genes, and that OTX015 specifically disrupts BRD4 binding and transcription of these genes. We show that OTX015 is effective against mouse and human MYCN-driven tumor models and that BRD4 not only targets MYCN, but specifically occupies MYCN target gene enhancers as well as other genes associated with super-enhancers. Clin Cancer Res; 22(10); 2470-81. ©2015 AACR. ©2015 American Association for Cancer Research.

The Homeodomain Transcription Factor Cdx1 Does Not Behave as an Oncogene in Normal Mouse Intestine1

PubMed Central

Crissey, Mary Ann S; Guo, Rong-Jun; Fogt, Franz; Li, Hong; Katz, Jonathan P; Silberg, Debra G; Suh, Eun Ran; Lynch, John P

2008-01-01

The Caudal-related homeobox genes Cdx1 and Cdx2 are intestine-specific transcription factors that regulate differentiation of intestinal cell types. Previously, we have shown Cdx1 to be antiproliferative and to promote cell differentiation. However, other studies have suggested that Cdx1 may be an oncogene. To test for oncogenic behavior, we used the murine villin promoter to ectopically express Cdx1 in the small intestinal villi and colonic surface epithelium. No changes in intestinal architecture, cell differentiation, or lineage selection were observed with expression of the transgene. Classic oncogenes enhance proliferation and induce tumors when ectopically expressed. However, the Cdx1 transgene neither altered intestinal proliferation nor induced spontaneous intestinal tumors. In a murine model for colitis-associated cancer, the Cdx1 transgene decreased, rather than increased, the number of adenomas that developed. In the polyps, the expression of the endogenous and the transgenic Cdx1 proteins was largely absent, whereas endogenous Villin expression was retained. This suggests that transgene silencing was specific and not due to a general Villin inactivation. In conclusion, neither the ectopic expression of Cdx1 was associated with changes in intestinal cell proliferation or differentiation nor was there increased intestinal cancer susceptibility. Our results therefore suggest that Cdx1 is not an oncogene in normal intestinal epithelium. PMID:18231635

miR-24-3p Suppresses Malignant Behavior of Lacrimal Adenoid Cystic Carcinoma by Targeting PRKCH to Regulate p53/p21 Pathway.

PubMed

Zhang, Ming-Xue; Zhang, Jie; Zhang, Hong; Tang, Hua

2016-01-01

MicroRNA (miRNA) may function as an oncogene or a tumor suppressor in tumorigenesis. However, the mechanism of miRNAs in adenoid cystic carcinoma (ACC) is unclear. Here, we provide evidence that miR-24-3p was downreglated and functions as a tumor suppressor in human lacrimal adenoid cystic carcinoma by suppressing proliferation and migration/invasion while promoting apoptosis. miR-24-3p down-regulated protein kinase C eta (PRKCH) by binding to its untranslated region (3'UTR). PRKCH increased the of the cell growth and migration/invasion in ACC cells and suppressed the expression of p53 and p21 in both mRNA and protein level. The overexpression of miR-24-3p decreased its malignant phenotype. Ectopic expression of PRKCH counteracted the suppression of malignancy induced by miR-24-3p, as well as ectopic expression of miR-24-3p rescued the suppression of PRKCH in the p53/p21 pathway. These results suggest that miR-24-3p promotes the p53/p21 pathway by down-regulating PRKCH expression in lacrimal adenoid cystic carcinoma cells.

Advancing osteochondral tissue engineering: bone morphogenetic protein, transforming growth factor, and fibroblast growth factor signaling drive ordered differentiation of periosteal cells resulting in stable cartilage and bone formation in vivo.

PubMed

Mendes, L F; Katagiri, H; Tam, W L; Chai, Y C; Geris, L; Roberts, S J; Luyten, F P

2018-02-21

Chondrogenic mesenchymal stem cells (MSCs) have not yet been used to address the clinical demands of large osteochondral joint surface defects. In this study, self-assembling tissue intermediates (TIs) derived from human periosteum-derived stem/progenitor cells (hPDCs) were generated and validated for stable cartilage formation in vivo using two different animal models. hPDCs were aggregated and cultured in the presence of a novel growth factor (GF) cocktail comprising of transforming growth factor (TGF)-β1, bone morphogenetic protein (BMP)2, growth differentiation factor (GDF)5, BMP6, and fibroblast growth factor (FGF)2. Quantitative polymerase chain reaction (PCR) and immunohistochemistry were used to study in vitro differentiation. Aggregates were then implanted ectopically in nude mice and orthotopically in critical-size osteochondral defects in nude rats and evaluated by microcomputed tomography (µCT) and immunohistochemistry. Gene expression analysis after 28 days of in vitro culture revealed the expression of early and late chondrogenic markers and a significant upregulation of NOGGIN as compared to human articular chondrocytes (hACs). Histological examination revealed a bilayered structure comprising of chondrocytes at different stages of maturity. Ectopically, TIs generated both bone and mineralized cartilage at 8 weeks after implantation. Osteochondral defects treated with TIs displayed glycosaminoglycan (GAG) production, type-II collagen, and lubricin expression. Immunostaining for human nuclei protein suggested that hPDCs contributed to both subchondral bone and articular cartilage repair. Our data indicate that in vitro derived osteochondral-like tissues can be generated from hPDCs, which are capable of producing bone and cartilage ectopically and behave orthotopically as osteochondral units.

Thiazolidinedione treatment and constitutive-PPARγ activation induces ectopic adipogenesis and promotes age-related thymic involution

PubMed Central

Youm, Yun-Hee; Yang, Hyunwon; Amin, Raj; Smith, Steven R.; Leff, Todd; Dixit, Vishwa Deep

2010-01-01

Age-related thymic involution is characterized by reduction in T cell production together with ectopic adipocyte development within the hematopoietic and thymic niches. PPARγ is required for adipocyte development, glucose homeostasis and is a target for several insulin-sensitizing drugs. Our prior studies showed that age-related elevation of PPARγ expression in thymic stromal cells is associated with thymic involution. Here, using clinically relevant pharmacological and genetic manipulations in mouse models, we provide evidence that activation of PPARγ leads to reduction in thymopoiesis. Treatment of aged mice with anti-hyperglycemic PPARγ-ligand class of Thiazolidinedione drug, Rosiglitazone caused robust thymic expression of classical pro-adipogenic transcripts. Rosiglitazone reduced thymic cellularity, lowered the naïve T cell number and T cell receptor excision circles (TRECs) indicative of compromised thymopoiesis. To directly investigate whether PPARγ activation induces thymic involution, we created transgenic mice with constitutive-active PPARγ (CA-PPARg) fusion protein in cells of adipogenic lineage. Importantly, CA-PPARγ transgene was expressed in thymus and in Fibroblast Specific Protein-1/S100A4 (FSP1+) cells, a marker of secondary mesenchymal cells. The CAPPARγ fusion protein mimicked the liganded PPARγ receptor and the transgenic mice displayed increased ectopic thymic adipogenesis and reduced thymopoiesis. Furthermore, the reduction in thymopoiesis in CA-PPARγ mice was associated with higher bone marrow adiposity and lower hematopoietic stem cell progenitor pool. Consistent with lower thymic output, CAPPARγ transgenic mice had restricted T cell receptor (TCR) repertoire diversity. Collectively, our data suggest that activation of PPARγ accelerates thymic aging and thymus-specific PPARγ antagonist may forestall age-related decline in T cell diversity. PMID:20374200

Downregulation of miRNA-30c and miR-203a is associated with hepatitis C virus core protein-induced epithelial–mesenchymal transition in normal hepatocytes and hepatocellular carcinoma cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu, Dongjing; Wu, Jilin, E-mail: 6296082@qq.com; Liu, Meizhou

Hepatitis C virus (HCV) Core protein has been demonstrated to induce epithelial–mesenchymal transition (EMT) and is associated with cancer progression of hepatocellular carcinoma (HCC). However, how the Core protein regulates EMT is still unclear. In this study, HCV Core protein was overexpressed by an adenovirus. The protein levels of EMT markers were measured by Western blot. The xenograft animal model was established by inoculation of HepG2 cells. Results showed that ectopic expression of HCV core protein induced EMT in L02 hepatocytes and HepG2 tumor cells by upregulating vimentin, Sanl1, and Snal2 expression and downregulating E-cadherin expression. Moreover, Core protein downregulatedmore » miR-30c and miR-203a levels in L02 and HepG2 cells, but artificial expression of miR-30c and miR-203a reversed Core protein-induced EMT. Further analysis showed that ectopic expression of HCV core protein stimulated cell proliferation, inhibited apoptosis, and increased cell migration, whereas artificial expression of miR-30c and miR-203a significantly reversed the role of Core protein in these cell functions in L02 and HepG2 cells. In the HepG2 xenograft tumor models, artificial expression of miR-30c and miR-203a inhibited EMT and tumor growth. Moreover, L02 cells overexpressing Core protein can form tumors in nude mice. In HCC patients, HCV infection significantly shortened patients' survival time, and loss of miR-30c and miR-203 expression correlated with poor survival. In conclusion, HCV core protein downregulates miR-30c and miR-203a expression, which results in activation of EMT in normal hepatocytes and HCC tumor cells. The Core protein-activated-EMT is involved in the carcinogenesis and progression of HCC. Loss of miR-30c and miR-203a expression is a marker for the poor prognosis of HCC. - Highlights: • HCV core protein downregulates miR-30c and miR-203a expression. • Downregulation of miR-30c and miR-203a activates EMT. • Activated-EMT is involved in the carcinogenesis and progression of HCC. • Loss of miR-30c and miR-203a expression is a marker for the poor prognosis of HCC.« less

The pepper cysteine/histidine-rich DC1 domain protein CaDC1 binds both RNA and DNA and is required for plant cell death and defense response.

PubMed

Hwang, In Sun; Choi, Du Seok; Kim, Nak Hyun; Kim, Dae Sung; Hwang, Byung Kook

2014-01-01

Plant defense against microbial pathogens is coordinated by a complex regulatory network. Cysteine/histidine-rich DC1 domain proteins mediate a variety of cellular processes involved in plant growth, development and stress responses. We identified a pepper (Capsicum annuum) cysteine/histidine-rich DC1 domain protein gene, CaDC1, which positively regulates plant defense during microbial infection, based on gene silencing and transient expression in pepper, as well as ectopic expression in Arabidopsis. Induction of CaDC1 by avirulent Xanthomonas campestris pv vesicatoria (Xcv) infection was pronounced at both transcriptional and translational levels in pepper leaves. Purified CaDC1 protein bound to both DNA and RNA in vitro, especially in the presence of Zn(2+). CaDC1 was localized to both the nucleus and the cytoplasm, which was required for plant cell death signaling. The nuclear localization of CaDC1 was dependent on the divergent C1 (DC1) domain. CaDC1 silencing in pepper conferred increased susceptibility to Xcv infection, which was accompanied by reduced salicylic acid accumulation and defense-related gene expression. Ectopic expression of CaDC1 in Arabidopsis enhanced resistance to Hyaloperonospora arabidopsidis. CaDC1 binds both RNA and DNA and functions as a positive regulator of plant cell death and SA-dependent defense responses. © 2013 The Authors. New Phytologist © 2013 New Phytologist Trust.

p62/SQSTM1 promotes rapid ubiquitin conjugation to target proteins after endosome rupture during xenophagy.

PubMed

Tsuchiya, Megumi; Ogawa, Hidesato; Koujin, Takako; Mori, Chie; Osakada, Hiroko; Kobayashi, Shouhei; Hiraoka, Yasushi; Haraguchi, Tokuko

2018-03-01

Autophagy is a bulk degradation pathway, and selective autophagy to remove foreign entities is called xenophagy. The conjugation of ubiquitin to target pathogens is an important process in xenophagy but when and where this ubiquitination occurs remains unclear. Here, we analyzed the temporal sequence and subcellular location of ubiquitination during xenophagy using time-lapse observations, with polystyrene beads mimicking invading pathogens. Results revealed accumulation of a ubiquitination marker around the beads within 3 min after endosome rupture. Recruitment of ubiquitin to the beads was significantly delayed in p62-knockout murine embryonic fibroblast cells, and this delay was rescued by ectopic p62 expression. Ectopic expression of a phosphorylation-mimicking p62 mutated at serine residue 405 (equivalent to human serine residue 403) rescued this delay, but its unphosphorylated form did not. These results indicate that ubiquitination mainly occurs after endosome rupture and suggest that p62, specifically the phosphorylated form, promotes ubiquitin conjugation to target proteins in xenophagy.

Evolutionary conservation of vertebrate notochord genes in the ascidian Ciona intestinalis.

PubMed

Kugler, Jamie E; Passamaneck, Yale J; Feldman, Taya G; Beh, Jeni; Regnier, Todd W; Di Gregorio, Anna

2008-11-01

To reconstruct a minimum complement of notochord genes evolutionarily conserved across chordates, we scanned the Ciona intestinalis genome using the sequences of 182 genes reported to be expressed in the notochord of different vertebrates and identified 139 candidate notochord genes. For 66 of these Ciona genes expression data were already available, hence we analyzed the expression of the remaining 73 genes and found notochord expression for 20. The predicted products of the newly identified notochord genes range from the transcription factors Ci-XBPa and Ci-miER1 to extracellular matrix proteins. We examined the expression of the newly identified notochord genes in embryos ectopically expressing Ciona Brachyury (Ci-Bra) and in embryos expressing a repressor form of this transcription factor in the notochord, and we found that while a subset of the genes examined are clearly responsive to Ci-Bra, other genes are not affected by alterations in its levels. We provide a first description of notochord genes that are not evidently influenced by the ectopic expression of Ci-Bra and we propose alternative regulatory mechanisms that might control their transcription. Copyright 2008 Wiley-Liss, Inc.

Optimized transitory ectopic expression of promastigote surface antigen protein in Nicotiana benthamiana, a potential anti-leishmaniasis vaccine candidate.

PubMed

Lacombe, Séverine; Bangratz, Martine; Brizard, Jean-Paul; Petitdidier, Elodie; Pagniez, Julie; Sérémé, Drissa; Lemesre, Jean-Loup; Brugidou, Christophe

2018-01-01

In recent years, plants have been shown to be an efficient alternative expression system for high-value pharmaceuticals such as vaccines. However, constitutive expression of recombinant protein remains uncertain on their level of production and biological activity. To overcome these problems, transitory expression systems have been developed. Here, a series of experiments were performed to determine the most effective conditions to enhance vaccine antigen transient accumulation in Nicotiana benthamiana leaves using the promastigote surface antigen (PSA) from the parasitic protozoan Leishmania infantum. This protein has been previously identified as the major antigen of a licensed canine anti-leishmaniasis vaccine. The classical prokaryote Escherichia coli biosystem failed in accumulating PSA. Consequently, the standard plant system based on N. benthamiana has been optimized for the production of putatively active PSA. First, the RNA silencing defense mechanism set up by the plant against PSA ectopic expression was abolished by using three viral suppressors acting at different steps of the RNA silencing pathway. Then, we demonstrated that the signal peptide at the N-terminal side of the PSA is required for its accumulation. The PSA ER signaling and retention with the PSA signal peptide and the KDEL motif, respectively were optimized to significantly increase its accumulation. Finally, we demonstrate that the production of recombinant PSA in N. benthamiana leaves allows the conservation of its immunogenic property. These approaches demonstrate that based on these optimizations, plant based systems can be used to effectively produce the biological active PSA protein. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Ectopic expression of a polyalanine expansion mutant of poly(A)-binding protein N1 in muscle cells in culture inhibits myogenesis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang Qishan; Bag, Jnanankur

2006-02-17

Oculopharyngeal muscular dystrophy (OPMD) is an adult-onset dominant genetic disease caused by the expansion of a GCG trinucleotide repeat that encodes the polyalanine tract at the N-terminus of the nuclear poly(A)-binding protein (PABPN1). Presence of intranuclear inclusions (INIs) containing PABPN1 aggregates in the skeletal muscles is the hallmark of OPMD. Here, we show that ectopic expression of the mutant PABPN1 produced INIs in a muscle cell culture model and reduced expression of several muscle-specific proteins including {alpha}-actin, slow troponin C, muscle creatine kinase, and two myogenic transcription factors, myogenin and MyoD. However, the levels of two upstream regulators of themore » MyoD gene, the Myf-5 and Pax3/7, were not affected, but both proteins co-localized with the PABPN1 aggregates in the mutant PABPN1 overexpressing cells. In these cells, although myogenin and MyoD levels were reduced, these two transcription factors did not co-localize with the mutant PABPN1 aggregates. Therefore, sequestration of Myf5 and Pax3/7 by the mutant PABPN1 aggregates was a specific effect on these factors. Our results suggest that trapping of these two important myogenic determinants may interfere with an early step in myogenesis.« less

Transcriptional deregulation of oncogenic myocyte enhancer factor 2C in T-cell acute lymphoblastic leukemia.

PubMed

Nagel, Stefan; Venturini, Letizia; Meyer, Corinna; Kaufmann, Maren; Scherr, Michaela; Drexler, Hans G; Macleod, Roderick A F

2011-02-01

Myocyte enhancer factor 2C (MEF2C) encodes a transcription factor which is ectopically expressed in T-cell acute lymphoblastic leukemia (T-ALL) cell lines, deregulated directly by ectopically expressed homeodomain protein NKX2-5 or by loss of promoter regions via del(5)(q14). Here, we analyzed the MEF2C 5'-region, thus identifying potential regulatory binding sites for GFI1B, basic helix-loop-helix proteins, STAT5, and HOXA9/HOXA10. Chromatin immunoprecipitation and overexpression analyses demonstrated direct activation by GFI1B and LYL1 and inhibition by STAT5. HOXA9/HOXA10 activated expression of NMYC which in turn mediated MEF2C repression, indicating an indirect mode of regulation via NMYC interactor (NMI) and STAT5. Lacking comma: Chromosomal deletion of the STAT5 binding site in LOUCY cells reduced protein levels of STAT5 in some MEF2C-positve T-ALL cell lines, and the presence of inhibitory IL7-JAK-STAT5 signaling highlighted the repressive impact of this factor in MEF2C regulation. Taken together, our results indicate that the expression of MEF2C in T-ALL cells is principally deregulated via activating leukemic transcription factors GFI1B or NKX2-5 and by escaping inhibitory developmental STAT5 signaling.

A novel fluorescent sensor for measurement of CFTR function by flow cytometry.

PubMed

Vijftigschild, Lodewijk A W; van der Ent, Cornelis K; Beekman, Jeffrey M

2013-06-01

Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene cause cystic fibrosis. CFTR-dependent iodide transport measured by fluorescent quenching of ectopically expressed halide-sensitive yellow fluorescent protein (YFP) is widely being used to study CFTR function by microscopy or plate readers. Since YFP fluorescence in these systems is dependent on YFP expression levels and iodide concentration, differences in sensor expression level between experimental units are normalized at the start of each experiment. To allow accurate measurement of CFTR function by flow cytometry, we reasoned that co-expression of an iodide insensitive fluorescent protein would allow for normalization of sensor expression levels and more accurate quantification of CFTR function. Our data indicated that dsRed and mKate fluorescence are iodide insensitive, and we determined an optimal format for co-expression of these fluorescent proteins with halide-sensitive YFP. We showed using microscopy that ratiometric measurement (YFP/mKate) corrects for differences in sensor expression levels. Ratiometric measurements were essential to accurately measure CFTR function by flow cytometry that we here describe for the first time. Mixing of wild type or mutant CFTR expressing cells indicated that addition of approximately 10% of wild type CFTR expressing cells could be distinguished by ratiometric YFP quenching. Flow cytometric ratiometric YFP quenching also allowed us to study CFTR mutants associated with differential residual function upon ectopic expression. Compared with conventional plate-bound CFTR function assays, the flow cytometric approach described here can be used to study CFTR function in suspension cells. It may be further adapted to study CFTR function in heterologous cell populations using cell surface markers and selection of cells that display high CFTR function by cell sorting. Copyright © 2013 International Society for Advancement of Cytometry.

The myogenic repressor gene Holes in muscles is a direct transcriptional target of Twist and Tinman in the Drosophila embryonic mesoderm.

PubMed

Elwell, Jennifer A; Lovato, TyAnna L; Adams, Melanie M; Baca, Erica M; Lee, Thai; Cripps, Richard M

2015-04-15

Understanding the regulatory circuitry controlling myogenesis is critical to understanding developmental mechanisms and developmentally-derived diseases. We analyzed the transcriptional regulation of a Drosophila myogenic repressor gene, Holes in muscles (Him). Previously, Him was shown to inhibit Myocyte enhancer factor-2 (MEF2) activity, and is expressed in myoblasts but not differentiating myotubes. We demonstrate that different phases of Him embryonic expression arises through the actions of different enhancers, and we characterize the enhancer required for its early mesoderm expression. This Him early mesoderm enhancer contains two conserved binding sites for the basic helix-loop-helix regulator Twist, and one binding site for the NK homeodomain protein Tinman. The sites for both proteins are required for enhancer activity in early embryos. Twist and Tinman activate the enhancer in tissue culture assays, and ectopic expression of either factor is sufficient to direct ectopic expression of a Him-lacZ reporter, or of the endogenous Him gene. Moreover, sustained expression of twist in the mesoderm up-regulates mesodermal Him expression in late embryos. Our findings provide a model to define mechanistically how Twist can both promotes myogenesis through direct activation of Mef2, and can place a brake on myogenesis, through direct activation of Him. Copyright © 2015 Elsevier Inc. All rights reserved.

Wnt/β-Catenin Regulates the Activity of Epiprofin/Sp6, SHH, FGF, and BMP to Coordinate the Stages of Odontogenesis

PubMed Central

Aurrekoetxea, Maitane; Irastorza, Igor; García-Gallastegui, Patricia; Jiménez-Rojo, Lucia; Nakamura, Takashi; Yamada, Yoshihiko; Ibarretxe, Gaskon; Unda, Fernando J.

2016-01-01

Background: We used an in vitro tooth development model to investigate the effects of overactivation of the Wnt/β-catenin pathway during odontogenesis by bromoindirubin oxime reagent (BIO), a specific inhibitor of GSK-3 activity. Results: Overactivating the Wnt/β-catenin pathway at tooth initiation upregulated and ectopically expressed the epithelial markers Sonic Hedgehog (Shh), Epiprofin (Epfn), and Fibroblast growth factor8 (Fgf8), which are involved in the delimitation of odontogenic fields in the oral ectoderm. This result indicated an ectopic extension of the odontogenic potential. During tooth morphogenesis, Fibroblast growth factor4 (Fgf4), Fibroblast growth factor10 (Fgf10), Muscle segment homeobox 1 (Msx-1), Bone Morphogenetic protein 4 (Bmp4), and Dickkopf WNT signaling pathway inhibitor 1 (Dkk-1) were overexpressed in first molars cultured with BIO. Conversely, the expression levels of Wingless integration site 10b (Wnt-10b) and Shh were reduced. Additionally, the odontoblast differentiation markers Nestin and Epfn showed ectopic overexpression in the dental mesenchyme of BIO-treated molars. Moreover, alkaline phosphatase activity increased in the dental mesenchyme, again suggesting aberrant, ectopic mesenchymal cell differentiation. Finally, Bmp4 downregulated Epfn expression during dental morphogenesis. Conclusions: We suggest the presence of a positive feedback loop wherein Epfn and β-catenin activate each other. The balance of the expression of these two molecules is essential for proper tooth development. We propose a possible link between Wnt, Bmp, and Epfn that would critically determine the correct patterning of dental cusps and the differentiation of odontoblasts and ameloblasts. PMID:27066482

The protein expression landscape of the Arabidopsis root

PubMed Central

Petricka, Jalean J.; Schauer, Monica A.; Megraw, Molly; Breakfield, Natalie W.; Thompson, J. Will; Georgiev, Stoyan; Soderblom, Erik J.; Ohler, Uwe; Moseley, Martin Arthur; Grossniklaus, Ueli; Benfey, Philip N.

2012-01-01

Because proteins are the major functional components of cells, knowledge of their cellular localization is crucial to gaining an understanding of the biology of multicellular organisms. We have generated a protein expression map of the Arabidopsis root providing the identity and cell type-specific localization of nearly 2,000 proteins. Grouping proteins into functional categories revealed unique cellular functions and identified cell type-specific biomarkers. Cellular colocalization provided support for numerous protein–protein interactions. With a binary comparison, we found that RNA and protein expression profiles are weakly correlated. We then performed peak integration at cell type-specific resolution and found an improved correlation with transcriptome data using continuous values. We performed GeLC-MS/MS (in-gel tryptic digestion followed by liquid chromatography-tandem mass spectrometry) proteomic experiments on mutants with ectopic and no root hairs, providing complementary proteomic data. Finally, among our root hair-specific proteins we identified two unique regulators of root hair development. PMID:22447775

The Circular RNA Interacts with STAT3, Increasing Its Nuclear Translocation and Wound Repair by Modulating Dnmt3a and miR-17 Function.

PubMed

Yang, Zhen-Guo; Awan, Faryal Mehwish; Du, William W; Zeng, Yan; Lyu, Juanjuan; Wu, De; Gupta, Shaan; Yang, Weining; Yang, Burton B

2017-09-06

Delayed or impaired wound healing is a major health issue worldwide, especially in patients with diabetes and atherosclerosis. Here we show that expression of the circular RNA circ-Amotl1 accelerated healing process in a mouse excisional wound model. Further studies showed that ectopic circ-Amotl1 increased protein levels of Stat3 and Dnmt3a. The increased Dnmt3a then methylated the promoter of microRNA miR-17, decreasing miR-17-5p levels but increasing fibronectin expression. We found that Stat3, similar to Dnmt3a and fibronectin, was a target of miR-17-5p. Decreased miR-17-5p levels would increase expression of fibronectin, Dnmt3a, and Stat3. All of these led to increased cell adhesion, migration, proliferation, survival, and wound repair. Furthermore, we found that circ-Amotl1 not only increased Stat3 expression but also facilitated Stat3 nuclear translocation. Thus, the ectopic expressed circ-Amotl1 and Stat3 were mainly translocated to nucleus. In the presence of circ-Amotl1, Stat3 interacted with Dnmt3a promoter with increased affinity, facilitating Dnmt3a transcription. Ectopic application of circ-Amotl1 accelerating wound repair may shed light on skin wound healing clinically. Copyright © 2017. Published by Elsevier Inc.

WEREWOLF and ENHANCER of GLABRA3 are interdependent regulators of the spatial expression pattern of GLABRA2 in Arabidopsis.

PubMed

Song, Sang-Kee; Kwak, Su-Hwan; Chang, Soo Chul; Schiefelbein, John; Lee, Myeong Min

2015-11-06

In multicellular organisms, cell fates are specified through differential regulation of transcription. Epidermal cell fates in the Arabidopsis thaliana root are precisely specified by several transcription factors, with the GLABRA2 (GL2) homeodomain protein acting at the farthest downstream in this process. To better understand the regulation of GL2 expression, we ectopically expressed WEREWOLF (WER) and ENHANCER OF GLABRA3 (EGL3) in various tissues and examined GL2 expression. Here we show that WER expressed ubiquitously in the root induced GL2 expression only in the root epidermis, whereas co-expression of WER and EGL3 induced GL2 expression in the corresponding tissues. We also found that GL3 accumulated in the nucleus at the early meristematic region and EGL3 accumulated later in the nucleus of epidermal cells. We further found that ectopic expression of WER and EGL3 in ground tissues inhibited GL2 expression in the epidermis. Our results suggest that the co-expression of WER and EGL3 is sufficient for driving GL2 and CPC expression. Copyright © 2015 Elsevier Inc. All rights reserved.

The enforced expression of c-Myc in pig fibroblasts triggers mesenchymal-epithelial transition (MET) via F-actin reorganization and RhoA/Rock pathway inactivation.

PubMed

Shi, Jun-Wen; Liu, Wei; Zhang, Ting-Ting; Wang, Sheng-Chun; Lin, Xiao-Lin; Li, Jing; Jia, Jun-Shuang; Sheng, Hong-Fen; Yao, Zhi-Fang; Zhao, Wen-Tao; Zhao, Zun-Lan; Xie, Rao-Ying; Yang, Sheng; Gao, Fei; Fan, Quan-Rong; Zhang, Meng-Ya; Yue, Min; Yuan, Jin; Gu, Wei-Wang; Yao, Kai-Tai; Xiao, Dong

2013-04-01

In previous studies from other labs it has been well demonstrated that the ectopic expression of c-Myc in mammary epithelial cells can induce epithelial-mesenchymal transition (EMT), whereas in our pilot experiment, epithelial-like morphological changes were unexpectedly observed in c-Myc-expressing pig fibroblasts [i.e., porcine embryonic fibroblasts (PEFs) and porcine dermal fibroblasts (PDFs)] and pig mesenchymal stem cells, suggesting that the same c-Myc gene is entitled to trigger EMT in epithelial cells and mesenchymal-epithelial transition (MET) in fibroblasts. This prompted us to characterize the existence of a MET in c-Myc-expressing PEFs and PDFs at the molecular level. qRT-PCR, immunofluorescence and western blot analysis illustrated that epithelial-like morphological changes were accompanied by the increased expression of epithelial markers [such as cell adhesion proteins (E-cadherin, α-catenin and Bves), tight junction protein occludin and cytokeratins (Krt8 and Krt18)], the reduced expression of mesenchymal markers [vimentin, fibronectin 1 (FN1), snail1, collagen family of proteins (COL1A1, COL5A2) and matrix metalloproteinase (MMP) family (MMP12 and MMP14)] and the decreased cell motility and increased cell adhesion in c-Myc-expressing PEFs and PDFs. Furthermore, the ectopic expression of c-Myc in pig fibroblasts disrupted the stress fiber network, suppressed the formation of filopodia and lamellipodia, and resulted in RhoA/Rock pathway inactivation, which finally participates in epithelial-like morphological conversion. Taken together, these findings demonstrate, for the first time, that the enforced expression of c-Myc in fibroblasts can trigger MET, to which cytoskeleton depolymerization and RhoA/Rock pathway inactivation contribute.

Enhanced epithelial to mesenchymal transition (EMT) and upregulated MYC in ectopic lesions contribute independently to endometriosis.

PubMed

Proestling, Katharina; Birner, Peter; Gamperl, Susanne; Nirtl, Nadine; Marton, Erika; Yerlikaya, Gülen; Wenzl, Rene; Streubel, Berthold; Husslein, Heinrich

2015-07-22

Epithelial to mesenchymal transition (EMT) is a process in which epithelial cells lose polarity and cell-to-cell contacts and acquire the migratory and invasive abilities of mesenchymal cells. These abilities are thought to be prerequisites for the establishment of endometriotic lesions. A hallmark of EMT is the functional loss of E-cadherin (CDH1) expression in epithelial cells. TWIST1, a transcription factor that represses E-cadherin transcription, is among the EMT inducers. SNAIL, a zinc-finger transcription factor, and its close relative SLUG have similar properties to TWIST1 and are thus also EMT inducers. MYC, which is upregulated by estrogens in the uterus by an estrogen response cis-acting element (ERE) in its promoter, is associated with proliferation in endometriosis. The role of EMT and proliferation in the pathogenesis of endometriosis was evaluated by analyzing TWIST1, CDH1 and MYC expression. CDH1, TWIST1, SNAIL and SLUG mRNA expression was analyzed by qRT-PCR from 47 controls and 74 patients with endometriosis. Approximately 42 ectopic and 62 eutopic endometrial tissues, of which 30 were matched samples, were collected during the same surgical procedure. We evaluated TWIST1 and MYC protein expression by immunohistochemistry (IHC) in the epithelial and stromal tissue of 69 eutopic and 90 ectopic endometrium samples, of which 49 matched samples were analyzed from the same patient. Concordant expression of TWIST1/SNAIL/SLUG and CDH1 but also of TWIST1 and MYC was analyzed. We found that TWIST1, SNAIL and SLUG are overexpressed (p < 0.001, p = 0.016 and p < 0.001) in endometriosis, while CDH1 expression was concordantly reduced in these samples (p < 0.001). Similar to TWIST1, the epithelial expression of MYC was also significantly enhanced in ectopic endometrium compared to eutopic tissues (p = 0.008). We found exclusive expression of either TWIST1 or MYC in the same samples (p = 0.003). Epithelial TWIST1 is overexpressed in endometriosis and may contribute to the formation of endometriotic lesions by inducing epithelial to mesenchymal transition, as CDH1 was reduced in ectopic lesions. We found exclusive expression of either TWIST1 or MYC in the same samples, indicating that EMT and proliferation contribute independently of each other to the formation of endometriotic lesions.

Activation of the Hedgehog Signaling Pathway in the Developing Lens Stimulates Ectopic FoxE3 Expression and Disruption in Fiber Cell Differentiation

PubMed Central

Kerr, Christine L.; Huang, Jian; Williams, Trevor; West-Mays, Judith A.

2012-01-01

Purpose. The signaling pathways and transcriptional effectors responsible for directing mammalian lens development provide key regulatory molecules that can inform our understanding of human eye defects. The hedgehog genes encode extracellular signaling proteins responsible for patterning and tissue formation during embryogenesis. Signal transduction of this pathway is mediated through activation of the transmembrane proteins smoothened and patched, stimulating downstream signaling resulting in the activation or repression of hedgehog target genes. Hedgehog signaling is implicated in eye development, and defects in hedgehog signaling components have been shown to result in defects of the retina, iris, and lens. Methods. We assessed the consequences of constitutive hedgehog signaling in the developing mouse lens using Cre-LoxP technology to express the conditional M2 smoothened allele in the embryonic head and lens ectoderm. Results. Although initial lens development appeared normal, morphological defects were apparent by E12.5 and became more significant at later stages of embryogenesis. Altered lens morphology correlated with ectopic expression of FoxE3, which encodes a critical gene required for human and mouse lens development. Later, inappropriate expression of the epithelial marker Pax6, and as well as fiber cell markers c-maf and Prox1 also occurred, indicating a failure of appropriate lens fiber cell differentiation accompanied by altered lens cell proliferation and cell death. Conclusions. Our findings demonstrate that the ectopic activation of downstream effectors of the hedgehog signaling pathway in the mouse lens disrupts normal fiber cell differentiation by a mechanism consistent with a sustained epithelial cellular developmental program driven by FoxE3. PMID:22491411

The Ras suppressor Rsu-1 binds to the LIM 5 domain of the adaptor protein PINCH1 and participates in adhesion-related functions

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dougherty, Gerard W.; Section on Structural Cell Biology, National Institute on Deafness and Communication Disorders; Chopp, Treasa

2005-05-15

Rsu-1 is a highly conserved leucine rich repeat (LRR) protein that is expressed ubiquitously in mammalian cells. Rsu-1 was identified based on its ability to inhibit transformation by Ras, and previous studies demonstrated that ectopic expression of Rsu-1 inhibited anchorage-independent growth of Ras-transformed cells and human tumor cell lines. Using GAL4-based yeast two-hybrid screening, the LIM domain protein, PINCH1, was identified as the binding partner of Rsu-1. PINCH1 is an adaptor protein that localizes to focal adhesions and it has been implicated in the regulation of adhesion functions. Subdomain mapping in yeast revealed that Rsu-1 binds to the LIM 5more » domain of PINCH1, a region not previously identified as a specific binding domain for any other protein. Additional testing demonstrated that PINCH2, which is highly homologous to PINCH1, except in the LIM 5 domain, does not interact with Rsu-1. Glutathione transferase fusion protein binding studies determined that the LRR region of Rsu-1 interacts with PINCH1. Transient expression studies using epitope-tagged Rsu-1 and PINCH1 revealed that Rsu-1 co-immunoprecipitated with PINCH1 and colocalized with vinculin at sites of focal adhesions in mammalian cells. In addition, endogenous P33 Rsu-1 from 293T cells co-immunoprecipitated with transiently expressed myc-tagged PINCH1. Furthermore, RNAi-induced reduction in Rsu-1 RNA and protein inhibited cell attachment, and while previous studies demonstrated that ectopic expression of Rsu-1 inhibited Jun kinase activation, the depletion of Rsu-1 resulted in activation of Jun and p38 stress kinases. These studies demonstrate that Rsu-1 interacts with PINCH1 in mammalian cells and functions, in part, by altering cell adhesion.« less

Ectopic expression of Arabidopsis broad-spectrum resistance gene RPW8.2 improves the resistance to powdery mildew in grapevine (Vitis vinifera).

PubMed

Hu, Yang; Li, Yajuan; Hou, Fengjuan; Wan, Dongyan; Cheng, Yuan; Han, Yongtao; Gao, Yurong; Liu, Jie; Guo, Ye; Xiao, Shunyuan; Wang, Yuejin; Wen, Ying-Qiang

2018-02-01

Powdery mildew is the most economically important disease of cultivated grapevines worldwide. Here, we report that the Arabidopsis broad-spectrum disease resistance gene RPW8.2 could improve resistance to powdery mildew in Vitis vinifera cv. Thompson Seedless. The RPW8.2-YFP fusion gene was stably expressed in grapevines from either the constitutive 35S promoter or the native promoter (NP) of RPW8.2. The grapevine shoots and plantlets transgenic for 35S::RPW8.2-YFP showed reduced rooting and reduced growth at later development stages in the absence of any pathogens. Infection tests with an adapted grapevine powdery mildew isolate En NAFU1 showed that hyphal growth and sporulation were significantly restricted in transgenic grapevines expressing either of the two constructs. The resistance appeared to be attributable to the ectopic expression of RPW8.2, and associated with the enhanced encasement of the haustorial complex (EHC) and onsite accumulation of H 2 O 2 . In addition, the RPW8.2-YFP fusion protein showed focal accumulation around the fungal penetration sites. Transcriptome analysis revealed that ectopic expression of RPW8.2 in grapevines not only significantly enhanced salicylic acid-dependent defense signaling, but also altered expression of other phytohormone-associated genes. Taken together, our results indicate that RPW8.2 could be utilized as a transgene for improving resistance against powdery mildew in grapevines. Copyright © 2017 Elsevier B.V. All rights reserved.

Regulation of Compound Leaf Development by PHANTASTICA in Medicago truncatula1[C][W][OPEN

PubMed Central

Ge, Liangfa; Peng, Jianling; Berbel, Ana; Madueño, Francisco; Chen, Rujin

2014-01-01

Plant leaves, simple or compound, initiate as peg-like structures from the peripheral zone of the shoot apical meristem, which requires class I KNOTTED-LIKE HOMEOBOXI (KNOXI) transcription factors to maintain its activity. The MYB domain protein encoded by the ASYMMETRIC LEAVES1/ROUGH SHEATH2/PHANTASTICA (ARP) gene, together with other factors, excludes KNOXI gene expression from incipient leaf primordia to initiate leaves and specify leaf adaxial identity. However, the regulatory relationship between ARP and KNOXI is more complex in compound-leafed species. Here, we investigated the role of ARP and KNOXI genes in compound leaf development in Medicago truncatula. We show that the M. truncatula phantastica mutant exhibited severe compound leaf defects, including curling and deep serration of leaf margins, shortened petioles, increased rachises, petioles acquiring motor organ characteristics, and ectopic development of petiolules. On the other hand, the M. truncatula brevipedicellus mutant did not exhibit visible compound leaf defects. Our analyses show that the altered petiole development requires ectopic expression of ELONGATED PETIOLULE1, which encodes a lateral organ boundary domain protein, and that the distal margin serration requires the auxin efflux protein M. truncatula PIN-FORMED10 in the M. truncatula phantastica mutant. PMID:24218492

Remote and reversible inhibition of neurons and circuits by small molecule induced potassium channel stabilization

PubMed Central

Auffenberg, Eva; Jurik, Angela; Mattusch, Corinna; Stoffel, Rainer; Genewsky, Andreas; Namendorf, Christian; Schmid, Roland M.; Rammes, Gerhard; Biel, Martin; Uhr, Manfred; Moosmang, Sven; Michalakis, Stylianos; Wotjak, Carsten T.; Thoeringer, Christoph K.

2016-01-01

Manipulating the function of neurons and circuits that translate electrical and chemical signals into behavior represents a major challenges in neuroscience. In addition to optogenetic methods using light-activatable channels, pharmacogenetic methods with ligand induced modulation of cell signaling and excitability have been developed. However, they are largely based on ectopic expression of exogenous or chimera proteins. Now, we describe the remote and reversible expression of a Kir2.1 type potassium channel using the chemogenetic technique of small molecule induced protein stabilization. Based on shield1-mediated shedding of a destabilizing domain fused to a protein of interest and inhibition of protein degradation, this principle has been adopted for biomedicine, but not in neuroscience so far. Here, we apply this chemogenetic approach in brain research for the first time in order to control a potassium channel in a remote and reversible manner. We could show that shield1-mediated ectopic Kir2.1 stabilization induces neuronal silencing in vitro and in vivo in the mouse brain. We also validated this novel pharmacogenetic method in different neurobehavioral paradigms.The DD-Kir2.1 may complement the existing portfolio of pharmaco- and optogenetic techniques for specific neuron manipulation, but it may also provide an example for future applications of this principle in neuroscience research. PMID:26757616

Ectopic Atoh1 expression drives Merkel cell production in embryonic, postnatal and adult mouse epidermis.

PubMed

Ostrowski, Stephen M; Wright, Margaret C; Bolock, Alexa M; Geng, Xuehui; Maricich, Stephen M

2015-07-15

Merkel cells are mechanosensitive skin cells whose production requires the basic helix-loop-helix transcription factor Atoh1. We induced ectopic Atoh1 expression in the skin of transgenic mice to determine whether Atoh1 was sufficient to create additional Merkel cells. In embryos, ectopic Atoh1 expression drove ectopic expression of the Merkel cell marker keratin 8 (K8) throughout the epidermis. Epidermal Atoh1 induction in adolescent mice similarly drove widespread K8 expression in glabrous skin of the paws, but in the whisker pads and body skin ectopic K8+ cells were confined to hair follicles and absent from interfollicular regions. Ectopic K8+ cells acquired several characteristics of mature Merkel cells in a time frame similar to that seen during postnatal development of normal Merkel cells. Although ectopic K8+ cell numbers decreased over time, small numbers of these cells remained in deep regions of body skin hair follicles at 3 months post-induction. In adult mice, greater numbers of ectopic K8+ cells were created by Atoh1 induction during anagen versus telogen and following disruption of Notch signaling by conditional deletion of Rbpj in the epidermis. Our data demonstrate that Atoh1 expression is sufficient to produce new Merkel cells in the epidermis, that epidermal cell competency to respond to Atoh1 varies by skin location, developmental age and hair cycle stage, and that the Notch pathway plays a key role in limiting epidermal cell competency to respond to Atoh1 expression. © 2015. Published by The Company of Biologists Ltd.

Involvement of calprotectin (S100A8/A9) in molecular pathways associated with HNSCC

PubMed Central

Khammanivong, Ali; Sorenson, Brent S.; Ross, Karen F.; Dickerson, Erin B.; Hasina, Rifat; Lingen, Mark W.; Herzberg, Mark C.

2016-01-01

Calprotectin (S100A8/A9), a heterodimeric protein complex of calcium-binding proteins S100A8 and S100A9, plays key roles in cell cycle regulation and inflammation, with potential functions in squamous cell differentiation. While upregulated in many cancers, S100A8/A9 is downregulated in squamous cell carcinomas of the cervix, esophagus, and the head and neck (HNSCC). We previously reported that ectopic S100A8/A9 expression inhibits cell cycle progression in carcinoma cells. Here, we show that declining expression of S100A8/A9 in patients with HNSCC is associated with increased DNA methylation, less differentiated tumors, and reduced overall survival. Upon ectopic over-expression of S100A8/A9, the cancer phenotype of S100A8/A9-negative carcinoma cells was suppressed in vitro and tumor growth in vivo was significantly decreased. MMP1, INHBA, FST, LAMC2, CCL3, SULF1, and SLC16A1 were significantly upregulated in HNSCC but were downregulated by S100A8/A9 expression. Our findings strongly suggest that downregulation of S100A8/A9 through epigenetic mechanisms may contribute to increased proliferation, malignant transformation, and disease progression in HNSCC. PMID:26883112

Involvement of calprotectin (S100A8/A9) in molecular pathways associated with HNSCC.

PubMed

Khammanivong, Ali; Sorenson, Brent S; Ross, Karen F; Dickerson, Erin B; Hasina, Rifat; Lingen, Mark W; Herzberg, Mark C

2016-03-22

Calprotectin (S100A8/A9), a heterodimeric protein complex of calcium-binding proteins S100A8 and S100A9, plays key roles in cell cycle regulation and inflammation, with potential functions in squamous cell differentiation. While upregulated in many cancers, S100A8/A9 is downregulated in squamous cell carcinomas of the cervix, esophagus, and the head and neck (HNSCC). We previously reported that ectopic S100A8/A9 expression inhibits cell cycle progression in carcinoma cells. Here, we show that declining expression of S100A8/A9 in patients with HNSCC is associated with increased DNA methylation, less differentiated tumors, and reduced overall survival. Upon ectopic over-expression of S100A8/A9, the cancer phenotype of S100A8/A9-negative carcinoma cells was suppressed in vitro and tumor growth in vivo was significantly decreased. MMP1, INHBA, FST, LAMC2, CCL3, SULF1, and SLC16A1 were significantly upregulated in HNSCC but were downregulated by S100A8/A9 expression. Our findings strongly suggest that downregulation of S100A8/A9 through epigenetic mechanisms may contribute to increased proliferation, malignant transformation, and disease progression in HNSCC.

Fine regulation of RhoA and Rock is required for skeletal muscle differentiation.

PubMed

Castellani, Loriana; Salvati, Erica; Alemà, Stefano; Falcone, Germana

2006-06-02

The RhoA GTPase controls a variety of cell functions such as cell motility, cell growth, and gene expression. Previous studies suggested that RhoA mediates signaling inputs that promote skeletal myogenic differentiation. We show here that levels and activity of RhoA protein are down-regulated in both primary avian myoblasts and mouse satellite cells undergoing differentiation, suggesting that a fine regulation of this GTPase is required. In addition, ectopic expression of activated RhoA in primary quail myocytes, but not in mouse myocytes, inhibits accumulation of muscle-specific proteins and cell fusion. By disrupting RhoA signaling with specific inhibitors, we have shown that this GTPase, although required for cell identity in proliferating myoblasts, is not essential for commitment to terminal differentiation and muscle gene expression. Ectopic expression of an activated form of its downstream effector, Rock, impairs differentiation of both avian and mouse myoblasts. Conversely, Rock inhibition with specific inhibitors and small interfering RNA-mediated gene silencing leads to accelerated progression in the lineage and enhanced cell fusion, underscoring a negative regulatory function of Rock in myogenesis. Finally, we have reported that Rock acts independently from RhoA in preventing myoblast exit from the cell cycle and commitment to differentiation and may receive signaling inputs from Raf-1 kinase.

Conditional and constitutive expression of a Tbx1-GFP fusion protein in mice

PubMed Central

2013-01-01

Background Velo-cardio-facial syndrome/DiGeorge syndrome (VCFS/DGS) is caused by a 1.5-3 Mb microdeletion of chromosome 22q11.2, frequently referred to as 22q11.2 deletion syndrome (22q11DS). This region includes TBX1, a T-box transcription factor gene that contributes to the etiology of 22q11DS. The requirement for TBX1 in mammalian development is dosage-sensitive, such that loss-of-function (LOF) and gain-of-function (GOF) of TBX1 in both mice and humans results in disease relevant congenital malformations. Results To further gain insight into the role of Tbx1 in development, we have targeted the Rosa26 locus to generate a new GOF mouse model in which a Tbx1-GFP fusion protein is expressed conditionally using the Cre/LoxP system. Tbx1-GFP expression is driven by the endogenous Rosa26 promoter resulting in ectopic and persistent expression. Tbx1 is pivotal for proper ear and heart development; ectopic activation of Tbx1-GFP in the otic vesicle by Pax2-Cre and Foxg1-Cre represses neurogenesis and produces morphological defects of the inner ear. Overexpression of a single copy of Tbx1-GFP using Tbx1Cre/+ was viable, while overexpression of both copies resulted in neonatal lethality with cardiac outflow tract defects. We have partially rescued inner ear and heart anomalies in Tbx1Cre/- null embryos by expression of Tbx1-GFP. Conclusions We have generated a new mouse model to conditionally overexpress a GFP-tagged Tbx1 protein in vivo. This provides a useful tool to investigate in vivo direct downstream targets and protein binding partners of Tbx1. PMID:23971992

Human Cytomegalovirus Nuclear Egress Proteins Ectopically Expressed in the Heterologous Environment of Plant Cells are Strictly Targeted to the Nuclear Envelope.

PubMed

Lamm, Christian E; Link, Katrin; Wagner, Sabrina; Milbradt, Jens; Marschall, Manfred; Sonnewald, Uwe

2016-03-10

In all eukaryotic cells, the nucleus forms a prominent cellular compartment containing the cell's nuclear genome. Although structurally similar, animal and plant nuclei differ substantially in details of their architecture. One example is the nuclear lamina, a layer of tightly interconnected filament proteins (lamins) underlying the nuclear envelope of metazoans. So far no orthologous lamin genes could be detected in plant genomes and putative lamin-like proteins are only poorly described in plants. To probe for potentially conserved features of metazoan and plant nuclear envelopes, we ectopically expressed the core nuclear egress proteins of human cytomegalovirus pUL50 and pUL53 in plant cells. pUL50 localizes to the inner envelope of metazoan nuclei and recruits the nuclear localized pUL53 to it, forming heterodimers. Upon expression in plant cells, a very similar localization pattern of both proteins could be determined. Notably, pUL50 is specifically targeted to the plant nuclear envelope in a rim-like fashion, a location to which coexpressed pUL53 becomes strictly corecruited from its initial nucleoplasmic distribution. Using pUL50 as bait in a yeast two-hybrid screening, the cytoplasmic re-initiation supporting protein RISP could be identified. Interaction of pUL50 and RISP could be confirmed by coexpression and coimmunoprecipitation in mammalian cells and by confocal laser scanning microscopy in plant cells, demonstrating partial pUL50-RISP colocalization in areas of the nuclear rim and other intracellular compartments. Thus, our study provides strong evidence for conserved structural features of plant and metazoan nuclear envelops and identifies RISP as a potential pUL50-interacting plant protein.

A physical interaction between viral replicase and capsid protein is required for genome-packaging specificity in an RNA virus.

PubMed

Seo, Jang-Kyun; Kwon, Sun-Jung; Rao, A L N

2012-06-01

Genome packaging is functionally coupled to replication in RNA viruses pathogenic to humans (Poliovirus), insects (Flock house virus [FHV]), and plants (Brome mosaic virus [BMV]). However, the underlying mechanism is not fully understood. We have observed previously that in FHV and BMV, unlike ectopically expressed capsid protein (CP), packaging specificity results from RNA encapsidation by CP that has been translated from mRNA produced from replicating genomic RNA. Consequently, we hypothesize that a physical interaction with replicase increases the CP specificity for packaging viral RNAs. We tested this hypothesis by evaluating the molecular interaction between replicase protein and CP using a FHV-Nicotiana benthamiana system. Bimolecular fluorescence complementation in conjunction with fluorescent cellular protein markers and coimmunoprecipitation assays demonstrated that FHV replicase (protein A) and CP physically interact at the mitochondrial site of replication and that this interaction requires the N-proximal region from either amino acids 1 to 31 or amino acids 32 to 50 of the CP. In contrast to the mitochondrial localization of CP derived from FHV replication, ectopic expression displayed a characteristic punctate pattern on the endoplasmic reticulum (ER). This pattern was altered to relocalize the CP throughout the cytoplasm when the C-proximal hydrophobic domain was deleted. Analysis of the packaging phenotypes of the CP mutants defective either in protein A-CP interactions or ER localization suggested that synchronization between protein A-CP interaction and its subcellular localization is imperative to confer packaging specificity.

A Physical Interaction between Viral Replicase and Capsid Protein Is Required for Genome-Packaging Specificity in an RNA Virus

PubMed Central

Seo, Jang-Kyun; Kwon, Sun-Jung

2012-01-01

Genome packaging is functionally coupled to replication in RNA viruses pathogenic to humans (Poliovirus), insects (Flock house virus [FHV]), and plants (Brome mosaic virus [BMV]). However, the underlying mechanism is not fully understood. We have observed previously that in FHV and BMV, unlike ectopically expressed capsid protein (CP), packaging specificity results from RNA encapsidation by CP that has been translated from mRNA produced from replicating genomic RNA. Consequently, we hypothesize that a physical interaction with replicase increases the CP specificity for packaging viral RNAs. We tested this hypothesis by evaluating the molecular interaction between replicase protein and CP using a FHV-Nicotiana benthamiana system. Bimolecular fluorescence complementation in conjunction with fluorescent cellular protein markers and coimmunoprecipitation assays demonstrated that FHV replicase (protein A) and CP physically interact at the mitochondrial site of replication and that this interaction requires the N-proximal region from either amino acids 1 to 31 or amino acids 32 to 50 of the CP. In contrast to the mitochondrial localization of CP derived from FHV replication, ectopic expression displayed a characteristic punctate pattern on the endoplasmic reticulum (ER). This pattern was altered to relocalize the CP throughout the cytoplasm when the C-proximal hydrophobic domain was deleted. Analysis of the packaging phenotypes of the CP mutants defective either in protein A-CP interactions or ER localization suggested that synchronization between protein A-CP interaction and its subcellular localization is imperative to confer packaging specificity. PMID:22438552

MAPK/ERK signal pathway involved expression of COX-2 and VEGF by IL-1β induced in human endometriosis stromal cells in vitro

PubMed Central

Huang, Fengying; Cao, Jing; Liu, Qiuhong; Zou, Ying; Li, Hongyun; Yin, Tuanfang

2013-01-01

Objective: Now there are more and more evidences that Cyclooxygenase-2 (COX-2) plays an important role in angiogenesis of endometriosis (EMs). Vascular endothelial growth factor (VEGF) has a potent angiogenic activity. However, it is worth studying about the regulating mechanism of COX-2/COX-1 and VEGF in the development of human endometriosis in vitro. The current study was designed to investigate the effect of 4 cytokines on COX-2/COX-1 expression and the effect of IL-1β on VEGF release in human endometriosis stromal cells (ESC), and to explore the related signaling pathways involved in vitro. Methods: Isolation, culture and identification of ESC. Cells were treated with 4 cytokines, and the inhibitor mitogen-activated protein-Erk (MEK) and the inhibitor p38 mitogen-activated protein kinase (MAPK) prior to adding cytokine IL-1β. COX-2 protein expression was measured by western blot and VEGF secretion was determined by ELISA. Results: Among four kinds of cytokines, IL-1β treatment increased COX-2 protein expression and VEGF release in three ESC, and TNF-α had the same effect on COX-2 protein level as IL-1β only in ectopic and eutopic ESC, and MCSF had only slight effect on ectopic ESC. In contrast, cytokines had no effect on COX-1 expression. We also demonstrated that MAPK reduced the synthesis of COX-2 by IL-1β induced. COX-2 inhibitor reduced VEGF release by IL-1β induced. Conclusions: i) In human ESC in vitro, IL-1β up-regulated the COX-2 expression through the activation of p38 MAPK pathway, and not to COX-1. ii) Up-regulation of VEGF level by IL-1β treatment was found in human endometriosis stromal cell and COX-2 inhibitor was involved in this process. PMID:24133591

Targeting of RNA Polymerase II by a nuclear Legionella pneumophila Dot/Icm effector SnpL.

PubMed

Schuelein, Ralf; Spencer, Hugh; Dagley, Laura F; Li, Peng Fei; Luo, Lin; Stow, Jennifer L; Abraham, Gilu; Naderer, Thomas; Gomez-Valero, Laura; Buchrieser, Carmen; Sugimoto, Chihiro; Yamagishi, Junya; Webb, Andrew I; Pasricha, Shivani; Hartland, Elizabeth L

2018-04-24

The intracellular pathogen Legionella pneumophila influences numerous eukaryotic cellular processes through the Dot/Icm-dependent translocation of more than 300 effector proteins into the host cell. Although many translocated effectors localize to the Legionella replicative vacuole, other effectors can affect remote intracellular sites. Following infection, a subset of effector proteins localizes to the nucleus where they subvert host cell transcriptional responses to infection. Here we identified Lpg2519 (Lpp2587/Lpw27461), as a new nuclear-localized effector that we have termed SnpL. Upon ectopic expression or during L. pneumophila infection, SnpL showed strong nuclear localization by immunofluorescence microscopy but was excluded from nucleoli. Using immunoprecipitation and mass spectrometry, we determined the host-binding partner of SnpL as the eukaryotic transcription elongation factor, SUPT5H/Spt5. SUPT5H is an evolutionarily conserved component of the DRB sensitivity-inducing factor complex (DSIF complex) that regulates RNA polymerase II (Pol II) dependent mRNA processing and transcription elongation. Protein interaction studies showed that SnpL bound to the central KOW motif region of SUPT5H. Ectopic expression of SnpL led to massive upregulation of host gene expression and macrophage cell death. The activity of SnpL further highlights the ability of L. pneumophila to control fundamental eukaryotic processes such as transcription that, in the case of SnpL, leads to global upregulation of host gene expression. This article is protected by copyright. All rights reserved.

Distal-less regulates eyespot patterns and melanization in Bicyclus butterflies.

PubMed

Monteiro, Antónia; Chen, Bin; Ramos, Diane M; Oliver, Jeffrey C; Tong, Xiaoling; Guo, Min; Wang, Wen-Kai; Fazzino, Lisa; Kamal, Firdous

2013-07-01

Butterfly eyespots represent novel complex traits that display substantial diversity in number and size within and across species. Correlative gene expression studies have implicated a large suite of transcription factors, including Distal-less (Dll), Engrailed (En), and Spalt (Sal), in eyespot development in butterflies, but direct evidence testing the function of any of these proteins is still missing. Here we show that the characteristic two-eyespot pattern of wildtype Bicyclus anynana forewings is correlated with dynamic progression of Dll, En, and Sal expression in larval wings from four spots to two spots, whereas no such decline in gene expression ensues in a four-eyespot mutant. We then conduct transgenic experiments testing whether over-expression of any of these genes in a wild-type genetic background is sufficient to induce eyespot differentiation in these pre-patterned wing compartments. We also produce a Dll-RNAi transgenic line to test how Dll down-regulation affects eyespot development. Finally we test how ectopic expression of these genes during the pupal stages of development alters adults color patters. We show that over-expressing Dll in larvae is sufficient to induce the differentiation of additional eyespots and increase the size of eyespots, whereas down-regulating Dll leads to a decrease in eyespot size. Furthermore, ectopic expression of Dll in the early pupal wing led to the appearance of ectopic patches of black scales. We conclude that Dll is a positive regulator of focal differentiation and eyespot signaling and that this gene is also a possible selector gene for scale melanization in butterflies. Copyright © 2013 Wiley Periodicals, Inc.

Aquaporin 5 Plays a Role in Estrogen-Induced Ectopic Implantation of Endometrial Stromal Cells in Endometriosis

PubMed Central

Jiang, Xiu Xiu; Fei, Xiang Wei; Zhao, Li; Ye, Xiao Lei; Xin, Liao Bin; Qu, Yang; Xu, Kai Hong; Wu, Rui Jin; Lin, Jun

2015-01-01

Aquaporin 5 (AQP5) participates in the migration of endometrial cells. Elucidation of the molecular mechanisms associated with AQP5-mediated, migration of endometrial cells may contribute to a better understanding of endometriosis. Our objectives included identifying the estrogen-response element (ERE) in the promoter region of the AQP5 gene, and, investigating the effects of AQP5 on ectopic implantation of endometrial cells. Luciferase reporter assays and electrophoretic mobility shift assay (EMSA) identified the ERE-like motif in the promoter region of the AQP5 gene. After blocking and up-regulating estradiol (E2) levels, we analysed the expression of AQP5 in endometrial stromal (ES) cells. After blocking E2 /or phosphatidylinositol 3 kinase(PI3K), we analysed the role of AQP5 in signaling pathways. We constructed an AQP5, shRNA, lentiviral vector to knock out the AQP5 gene in ES cells. After knock-out of the AQP5 gene, we studied the role of AQP5 in cell invasion, proliferation, and the formation of ectopic endometrial implants in female mice. We identified an estrogen-response element in the promoter region of the AQP5 gene. Estradiol (E2) increased AQP5 expression in a dose-dependent fashion, that was blocked by ICI182,780(an estrogen receptor inhibitor). E2 activated PI3K /protein kinase B(AKT) pathway (PI3K/AKT), that, in turn, increased AQP5 expression. LY294002(PI3K inhibitor) attenuated estrogen-enhanced, AQP5 expression. Knock-out of the AQP5 gene with AQP5 shRNA lentiviral vector significantly inhibited E2-enhanced invasion, proliferation of ES cells and formation of ectopic implants. Estrogen induces AQP5 expression by activating ERE in the promoter region of the AQP5gene, activates the PI3K/AKT pathway, and, promotes endometrial cell invasion and proliferation. These results provide new insights into some of the mechanisms that may underpin the development of deposits of ectopic endometrium. PMID:26679484

ABA Suppresses Root Hair Growth via the OBP4 Transcriptional Regulator1[OPEN

PubMed Central

Kawamura, Ayako; Schäfer, Sabine; Breuer, Christian; Shibata, Michitaro; Mitsuda, Nobutaka; Ohme-Takagi, Masaru; Matsui, Minami

2017-01-01

Plants modify organ growth and tune morphogenesis in response to various endogenous and environmental cues. At the cellular level, organ growth is often adjusted by alterations in cell growth, but the molecular mechanisms underlying this control remain poorly understood. In this study, we identify the DNA BINDING WITH ONE FINGER (DOF)-type transcription regulator OBF BINDING PROTEIN4 (OBP4) as a repressor of cell growth. Ectopic expression of OBP4 in Arabidopsis (Arabidopsis thaliana) inhibits cell growth, resulting in severe dwarfism and the repression of genes involved in the regulation of water transport, root hair development, and stress responses. Among the basic helix-loop-helix transcription factors known to control root hair growth, OBP4 binds the ROOT HAIR DEFECTIVE6-LIKE2 (RSL2) promoter to repress its expression. The accumulation of OBP4 proteins is detected in expanding root epidermal cells, and its expression level is increased by the application of abscisic acid (ABA) at concentrations sufficient to inhibit root hair growth. ABA-dependent induction of OBP4 is associated with the reduced expression of RSL2. Furthermore, ectopic expression of OBP4 or loss of RSL2 function results in ABA-insensitive root hair growth. Taken together, our results suggest that OBP4-mediated transcriptional repression of RSL2 contributes to the ABA-dependent inhibition of root hair growth in Arabidopsis. PMID:28167701

Molecular cloning and functional analysis of the FLOWERING LOCUS T (FT) homolog GhFT1 from Gossypium hirsutum.

PubMed

Guo, Danli; Li, Chao; Dong, Rui; Li, Xiaobo; Xiao, Xiangwen; Huang, Xianzhong

2015-06-01

FLOWERING LOCUS T (FT) encodes a member of the phosphatidylethanolamine-binding protein (PEBP) family that functions as the mobile floral signal, playing an important role in regulating the floral transition in angiosperms. We isolated an FT-homolog (GhFT1) from Gossypium hirsutum L. cultivar, Xinluzao 33 GhFT1 was predominantly expressed in stamens and sepals, and had a relatively higher expression level during the initiation stage of fiber development. GhFT1 mRNA displayed diurnal oscillations in both long-day and short-day condition, suggesting that the expression of this gene may be under the control of the circadian clock. Subcellular analysis revealed that GhFT1 protein located in the cytoplasm and nucleus. Ectopic expression of GhFT1 in transgenic arabidopsis plants resulted in early flowering compared with wild-type plants. In addition, ectopic expression of GhFT1 in arabidopsis ft-10 mutants partially rescued the extremely late flowering phenotype. Finally, several flowering related genes functioning downstream of AtFT were highly upregulated in the 35S::GhFT1 transgenic arabidopsis plants. In summary, GhFT1 is an FT-homologous gene in cotton that regulates flower transition similar to its orthologs in other plant species and thus it may be a candidate target for promoting early maturation in cotton breeding. © 2014 Institute of Botany, Chinese Academy of Sciences.

Regulatory role of AINTEGUMENTA in organ initiation and growth

DOE Office of Scientific and Technical Information (OSTI.GOV)

Krizek, Beth Allyn; Lebioda, Lukasz

2005-03-01

Although several members of the plant-specific AP2/ERF family of transcription factors are important developmental regulators, many genes in this large protein family remain uncharacterized. Here, we present a phylogenetic analysis of the18 genes that make up the AP2 subgroup of this family. We report expression analyses of seven Arabidopsis genes most closely related to the floral development gene AINTEGUMENTA and show that all AINTEGUMENTA-like (AIL) genes are transcribed in multiple tissues during development. They are expressed primarily in young actively dividing tissues of a plant and not in mature leaves or stems. The spatial distribution of AIL5, AIL6, and AIL7more » mRNA in inflorescences was characterized by in situ hybridization. Each of these genes is expressed in a spatially and temporally distinct pattern within inflorescence meristems and flowers. Ectopic expression of AIL5 resulted in a larger floral organ phenotype, similar to that resulting from ectopic expression of ANT. Our results are consistent with AIL genes having roles in specification of meristematic or division-competent states.« less

Tendon Mineralization Is Progressive and Associated with Deterioration of Tendon Biomechanical Properties, and Requires BMP-Smad Signaling in the Mouse Achilles Tendon Injury Model

PubMed Central

Zhang, Kairui; Asai, Shuji; Hast, Michael W.; Liu, Min; Usami, Yu; Iwamoto, Masahiro; Soslowsky, Louis J.; Enomoto-Iwamoto, Motomi

2016-01-01

Ectopic tendon mineralization can develop following tendon rupture or trauma surgery. The pathogenesis of ectopic tendon mineralization and its clinical impact have not been fully elucidated yet. In this study, we utilized a mouse Achilles tendon injury model to determine whether ectopic tendon mineralization alters the biomechanical properties of the tendon and whether BMP signaling is involved in this condition. A complete transverse incision was made at the midpoint of the right Achilles tendon in 8-week-old CD1 mice and the gap was left open. Ectopic cartilaginous mass formation was found in the injured tendon by 4 weeks post-surgery and ectopic mineralization was detected at 8–10 weeks post-surgery. Ectopic mineralization grew over time and volume of the mineralized materials of 25-weeks samples was about 2.5 fold bigger than that of 10-weeks samples, indicating that injury-induced ectopic tendon mineralization is progressive. In vitro mechanical testing showed that max force, max stress and mid-substance modulus in the 25-weeks samples were significantly lower than the 10-weeks samples. We observed substantial increases in expression of bone morphogenetic protein family genes in injured tendons 1 week post-surgery. Immunohistochemical analysis showed that phosphorylation of both Smad1 and Smad3 were highly increased in injured tendons as early as 1 week post-injury and remained high in ectopic chondrogenic lesions 4 weeks post-injury. Treatment with the BMP receptor kinase inhibitor (LDN193189) significantly inhibited injury-induced tendon mineralization. These findings indicate that injury-induced ectopic tendon mineralization is progressive, involves BMP signaling and associated with deterioration of tendon biomechanical properties. PMID:26825318

Curcumin Significantly Enhances Dual PI3K/Akt and mTOR Inhibitor NVP-BEZ235-Induced Apoptosis in Human Renal Carcinoma Caki Cells through Down-Regulation of p53-Dependent Bcl-2 Expression and Inhibition of Mcl-1 Protein Stability

PubMed Central

Cho, Il Je; Kim, Sang Chan; Kwon, Taeg Kyu

2014-01-01

The PI3K/Akt and mTOR signaling pathways are important for cell survival and growth, and they are highly activated in cancer cells compared with normal cells. Therefore, these signaling pathways are targets for inducing cancer cell death. The dual PI3K/Akt and mTOR inhibitor NVP-BEZ235 completely inhibited both signaling pathways. However, NVP-BEZ235 had no effect on cell death in human renal carcinoma Caki cells. We tested whether combined treatment with natural compounds and NVP-BEZ235 could induce cell death. Among several chemopreventive agents, curcumin, a natural biologically active compound that is extracted from the rhizomes of Curcuma species, markedly induced apoptosis in NVP-BEZ235-treated cells. Co-treatment with curcumin and NVP-BEZ235 led to the down-regulation of Mcl-1 protein expression but not mRNA expression. Ectopic expression of Mcl-1 completely inhibited curcumin plus NVP-NEZ235-induced apoptosis. Furthermore, the down-regulation of Bcl-2 was involved in curcumin plus NVP-BEZ235-induced apoptosis. Curcumin or NVP-BEZ235 alone did not change Bcl-2 mRNA or protein expression, but co-treatment reduced Bcl-2 mRNA and protein expression. Combined treatment with NVP-BEZ235 and curcumin reduced Bcl-2 expression in wild-type p53 HCT116 human colon carcinoma cells but not p53-null HCT116 cells. Moreover, Bcl-2 expression was completely reversed by treatment with pifithrin-α, a p53-specific inhibitor. Ectopic expression of Bcl-2 also inhibited apoptosis in NVP-BE235 plus curcumin-treated cells. In contrast, NVP-BEZ235 combined with curcumin did not have a synergistic effect on normal human skin fibroblasts and normal human mesangial cells. Taken together, combined treatment with NVP-BEZ235 and curcumin induces apoptosis through p53-dependent Bcl-2 mRNA down-regulation at the transcriptional level and Mcl-1 protein down-regulation at the post-transcriptional level. PMID:24743574

The Gem GTP-binding protein promotes morphological differentiation in neuroblastoma.

PubMed

Leone, A; Mitsiades, N; Ward, Y; Spinelli, B; Poulaki, V; Tsokos, M; Kelly, K

2001-05-31

Gem is a small GTP-binding protein within the Ras superfamily whose function has not been determined. We report here that ectopic Gem expression is sufficient to stimulate cell flattening and neurite extension in N1E-115 and SH-SY5Y neuroblastoma cells, suggesting a role for Gem in cytoskeletal rearrangement and/or morphological differentiation of neurons. Consistent with this potential function, in clinical samples of neuroblastoma, Gem protein was most highly expressed within cells which had differentiated to express ganglionic morphology. Gem was also observed in developing trigeminal nerve ganglia in 12.5 day mouse embryos, demonstrating that Gem expression is a property of normal ganglionic development. Although Gem expression is rare in epithelial and hematopoietic cancer cell lines, constitutive Gem levels were detected in several neuroblastoma cell lines and could be further induced as much as 10-fold following treatment with PMA or the acetylcholine muscarinic agonist, carbachol.

Molecular analysis of the mouse agouti gene and the role of dominant agouti-locus mutations in obesity and insulin resistance

DOE Office of Scientific and Technical Information (OSTI.GOV)

Klebig, M.L.; Woychik, R.P.; Wilkinson, J.E.

1994-09-01

The lethal yellow (A{sup y/-}) and viable yellow (A{sup vy/-}) mouse agouti mutants have a predominantly yellow pelage and display a complex syndrome that includes obesity, hyperinsulinemia, and insulin resistance, hallmark features of obesity-associated noninsulin-dependent diabetes mellitus (NIDDM) in humans. A new dominant agouti allele, A{sup iapy}, has recently been identified; like the A{sup vy} allele, it is homozygous viable and confers obesity and yellow fur in heterozygotes. The agouti gene was cloned and characterized at the molecular level. The gene is expressed in the skin during hair growth and is predicted to encode a 131 amino acid protein, thatmore » is likely to be a secreted factor. In both Ay/- and A{sup iapy}/- mice, the obesity and other dominant pleiotropic effects are associated with an ectopic expression of agouti in many tissues where the gene product is normally not produced. In Ay, a 170-kb deletion has occurred that causes an upstream promoter to drive the ectopic expression of the wild-type agouti coding exons. In A{sup iapy}, the coding region of the gene is expressed from a cryptic promoter within the LTR of an intracisternal A-particle (IAP), which has integrated within the region just upstream of the first agouti coding exon. Transgenic mice ubiquitously expressing the cloned agouti gene under the influence of the beta-actin and phosphoglycerate kinase promoters display obesity, hyperinsulinemia, and yellow coat color. This demonstrates unequivocally that ectopic expression of agouti is responsible for the yellow obese syndrome.« less

Ectopic KNOX Expression Affects Plant Development by Altering Tissue Cell Polarity and Identity[OPEN

PubMed Central

Rebocho, Alexandra B.

2016-01-01

Plant development involves two polarity types: tissue cell (asymmetries within cells are coordinated across tissues) and regional (identities vary spatially across tissues) polarity. Both appear altered in the barley (Hordeum vulgare) Hooded mutant, in which ectopic expression of the KNOTTED1-like Homeobox (KNOX) gene, BKn3, causes inverted polarity of differentiated hairs and ectopic flowers, in addition to wing-shaped outgrowths. These lemma-specific effects allow the spatiotemporal analysis of events following ectopic BKn3 expression, determining the relationship between KNOXs, polarity, and shape. We show that tissue cell polarity, based on localization of the auxin transporter SISTER OF PINFORMED1 (SoPIN1), dynamically reorients as ectopic BKn3 expression increases. Concurrently, ectopic expression of the auxin importer LIKE AUX1 and boundary gene NO APICAL MERISTEM is activated. The polarity of hairs reflects SoPIN1 patterns, suggesting that tissue cell polarity underpins oriented cell differentiation. Wing cell files reveal an anisotropic growth pattern, and computational modeling shows how polarity guiding growth can account for this pattern and wing emergence. The inverted ectopic flower orientation does not correlate with SoPIN1, suggesting that this form of regional polarity is not controlled by tissue cell polarity. Overall, the results suggest that KNOXs trigger different morphogenetic effects through interplay between tissue cell polarity, identity, and growth. PMID:27553356

Progression of neuronal and synaptic remodeling in the rd10 mouse model of retinitis pigmentosa.

PubMed

Phillips, M Joseph; Otteson, Deborah C; Sherry, David M

2010-06-01

The Pde6b(rd10) (rd10) mouse has a moderate rate of photoreceptor degeneration and serves as a valuable model for human autosomal recessive retinitis pigmentosa (RP). We evaluated the progression of neuronal remodeling of second- and third-order retinal cells and their synaptic terminals in retinas from Pde6b(rd10) (rd10) mice at varying stages of degeneration ranging from postnatal day 30 (P30) to postnatal month 9.5 (PNM9.5) using immunolabeling for well-known cell- and synapse-specific markers. Following photoreceptor loss, changes occurred progressively from outer to inner retina. Horizontal cells and rod and cone bipolar cells underwent morphological remodeling that included loss of dendrites, cell body migration, and the sprouting of ectopic processes. Gliosis, characterized by translocation of Müller cell bodies to the outer retina and thickening of their processes, was evident by P30 and became more pronounced as degeneration progressed. Following rod degeneration, continued expression of VGluT1 in the outer retina was associated with survival and expression of synaptic proteins by nearby second-order neurons. Rod bipolar cell terminals showed a progressive reduction in size and ectopic bipolar cell processes extended into the inner nuclear layer and ganglion cell layer by PNM3.5. Putative ectopic conventional synapses, likely arising from amacrine cells, were present in the inner nuclear layer by PNM9.5. Despite these changes, the laminar organization of bipolar and amacrine cells and the ON-OFF organization in the inner plexiform layer was largely preserved. Surviving cone and bipolar cell terminals continued to express the appropriate cell-specific presynaptic proteins needed for synaptic function up to PNM9.5. (c) 2010 Wiley-Liss, Inc.

Progression of Neuronal and Synaptic Remodeling in the rd10 Mouse Model of Retinitis Pigmentosa

PubMed Central

Phillips, M. Joseph; Otteson, Deborah C.; Sherry, David M.

2010-01-01

The Pde6brd10 (rd10) mouse has a moderate rate of photoreceptor degeneration and serves as a valuable model for human autosomal recessive retinitis pigmentosa (RP). We evaluated the progression of neuronal remodeling of second- and third-order retinal cells and their synaptic terminals in retinas from Pde6brd10 (rd10) mice at varying stages of degeneration ranging from postnatal day 30 (P30) to postnatal month 9.5 (PNM9.5) using immunolabeling for well known cell- and synapse-specific markers. Following photoreceptor loss, changes occurred progressively from outer to inner retina. Horizontal cells and rod and cone bipolar cells underwent morphological remodeling that included loss of dendrites, cell body migration, and the sprouting of ectopic processes. Gliosis, characterized by translocation of Müller cell bodies to the outer retina and thickening of their processes, was evident by P30 and became more pronounced as degeneration progressed. Following rod degeneration, continued expression of VGluT1 in the outer retina was associated with survival and expression of synaptic proteins by nearby second-order neurons. Rod bipolar cell terminals showed a progressive reduction in size and ectopic bipolar cell processes extended into the inner nuclear layer and ganglion cell layer by PNM3.5. Putative ectopic conventional synapses, likely arising from amacrine cells, were present in the inner nuclear layer by PNM9.5. Despite these changes, the laminar organization of bipolar and amacrine cells and the ON-OFF organization in the inner plexiform layer was largely preserved. Surviving cone and bipolar cell terminals continued to express the appropriate cell-specific presynaptic proteins needed for synaptic function up to PNM9.5. PMID:20394059

The Role of SncN and Ski in Mammary Epithelial Cell Transformation

DTIC Science & Technology

2005-07-01

cellular activities through the Smad proteins. Upon phosphorylation by the active TGFβ receptor kinases, Smad2 and Smad3 oligomerize with Smad4, translocate...sequence in sense and antisense orientation with an intervening linker . Primer pairs were designed to generate single-strand overhangs upon annealing...nuclear protein, based on studies of ectopically expressed SnoN and endogenous SnoN in cancer cell lines. In the nucleus, SnoN binds to Smad2, Smad3 , and

Gla-Rich Protein Is a Potential New Vitamin K Target in Cancer: Evidences for a Direct GRP-Mineral Interaction

PubMed Central

Viegas, Carla S. B.; Herfs, Marjolein; Rafael, Marta S.; Enriquez, José L.; Teixeira, Alexandra; Luís, Inês M.; van ‘t Hoofd, Cynthia M. R.; João, Alexandre; Maria, Vera L.; Cavaco, Sofia; Ferreira, Ana; Serra, Manuel; Theuwissen, Elke; Vermeer, Cees; Simes, Dina C.

2014-01-01

Gla-rich protein (GRP) was described in sturgeon as a new vitamin-K-dependent protein (VKDP) with a high density of Gla residues and associated with ectopic calcifications in humans. Although VKDPs function has been related with γ-carboxylation, the Gla status of GRP in humans is still unknown. Here, we investigated the expression of recently identified GRP spliced transcripts, the γ-carboxylation status, and its association with ectopic calcifications, in skin basal cell and breast carcinomas. GRP-F1 was identified as the predominant splice variant expressed in healthy and cancer tissues. Patterns of γ-carboxylated GRP (cGRP)/undercarboxylated GRP (ucGRP) accumulation in healthy and cancer tissues were determined by immunohistochemistry, using newly developed conformation-specific antibodies. Both GRP protein forms were found colocalized in healthy tissues, while ucGRP was the predominant form associated with tumor cells. Both cGRP and ucGRP found at sites of microcalcifications were shown to have in vitro calcium mineral-binding capacity. The decreased levels of cGRP and predominance of ucGRP in tumor cells suggest that GRP may represent a new target for the anticancer potential of vitamin K. Also, the direct interaction of cGRP and ucGRP with BCP crystals provides a possible mechanism explaining GRP association with pathological mineralization. PMID:24949434

Positive and Negative Regulation of Muscle Cell Identity by Members of the hedgehog and TGF-β Gene Families

PubMed Central

Du, Shao Jun; Devoto, Stephen H.; Westerfield, Monte; Moon, Randall T.

1997-01-01

We have examined whether the development of embryonic muscle fiber type is regulated by competing influences between Hedgehog and TGF-β signals, as previously shown for development of neuronal cell identity in the neural tube. We found that ectopic expression of Hedgehogs or inhibition of protein kinase A in zebrafish embryos induces slow muscle precursors throughout the somite but muscle pioneer cells only in the middle of the somite. Ectopic expression in the notochord of Dorsalin-1, a member of the TGF-β superfamily, inhibits the formation of muscle pioneer cells, demonstrating that TGF-β signals can antagonize the induction of muscle pioneer cells by Hedgehog. We propose that a Hedgehog signal first induces the formation of slow muscle precursor cells, and subsequent Hedgehog and TGF-β signals exert competing positive and negative influences on the development of muscle pioneer cells. PMID:9314535

G protein-coupled odorant receptors underlie mechanosensitivity in mammalian olfactory sensory neurons

PubMed Central

Connelly, Timothy; Yu, Yiqun; Grosmaitre, Xavier; Wang, Jue; Santarelli, Lindsey C.; Savigner, Agnes; Qiao, Xin; Wang, Zhenshan; Storm, Daniel R.; Ma, Minghong

2015-01-01

Mechanosensitive cells are essential for organisms to sense the external and internal environments, and a variety of molecules have been implicated as mechanical sensors. Here we report that odorant receptors (ORs), a large family of G protein-coupled receptors, underlie the responses to both chemical and mechanical stimuli in mouse olfactory sensory neurons (OSNs). Genetic ablation of key signaling proteins in odor transduction or disruption of OR–G protein coupling eliminates mechanical responses. Curiously, OSNs expressing different OR types display significantly different responses to mechanical stimuli. Genetic swap of putatively mechanosensitive ORs abolishes or reduces mechanical responses of OSNs. Furthermore, ectopic expression of an OR restores mechanosensitivity in loss-of-function OSNs. Lastly, heterologous expression of an OR confers mechanosensitivity to its host cells. These results indicate that certain ORs are both necessary and sufficient to cause mechanical responses, revealing a previously unidentified mechanism for mechanotransduction. PMID:25550517

Circular RNA HIPK3 promotes gallbladder cancer cell growth by sponging microRNA-124.

PubMed

Kai, Ding; Yannian, Liao; Yitian, Chen; Dinghao, Gong; Xin, Zhao; Wu, Ji

2018-06-18

Recent studies have implied that circHIPK3, an abundant circular RNA (circRNA), participates in tumorigenesis and cancer progression. Its expression and potential functions in human gallbladder cancer were examined in this study. We show that circHIPK3 is upregulated in human gallbladder cancer cells. But its level is low in gallbladder epithelial cells. circHIPK3 silencing by targeted siRNA potently inhibited survival and proliferation of established and primary human gallbladder cancer cells, while inducing cell apoptosis. Conversely, ectopic over-expression of circHIPK3 can further promote cancer cell proliferation. In gallbladder cancer cells, circHIPK3 sponged the tumor-suppressive microRNA-124 (miR-124) to sequester and inhibit its activity, thereby leading to increased expression of miR-124 targets, including ROCK1 (rho-associated protein kinase 1) and CDK6 (rho-associated protein kinase). Ectopic over-expression of miR-124 b y a lentiviral vector mimicked and abolished actions by circHIPK3 siRNA in gallbladder cancer cells. At last, we show that circHIPK3 is upregulated in human gallbladder cancer tissues, which is correlated with miR-124 downregulation and ROCK1-CDK6 upregulation. Together, we conclude that circHIPK3 promotes gallbladder cancer cell growth possibly by sponging miR-124. The over-expressed circHIPK3 could be a novel therapeutic target and diagnosis marker of human gallbladder cancer. Copyright © 2018. Published by Elsevier Inc.

OXPHOS-Mediated Induction of NAD+ Promotes Complete Oxidation of Fatty Acids and Interdicts Non-Alcoholic Fatty Liver Disease.

PubMed

Akie, Thomas E; Liu, Lijun; Nam, Minwoo; Lei, Shi; Cooper, Marcus P

2015-01-01

OXPHOS is believed to play an important role in non-alcoholic fatty liver disease (NAFLD), however, precise mechanisms whereby OXPHOS influences lipid homeostasis are incompletely understood. We previously reported that ectopic expression of LRPPRC, a protein that increases cristae density and OXPHOS, promoted fatty acid oxidation in cultured primary hepatocytes. To determine the biological significance of that observation and define underlying mechanisms, we have ectopically expressed LRPPRC in mouse liver in the setting of NAFLD. Interestingly, ectopic expression of LRPPRC in mouse liver completely interdicted NAFLD, including inflammation. Consistent with mitigation of NAFLD, two markers of hepatic insulin resistance--ROS and PKCε activity--were both modestly reduced. As reported by others, improvement of NAFLD was associated with improved whole-body insulin sensitivity. Regarding hepatic lipid homeostasis, the ratio of NAD+ to NADH was dramatically increased in mouse liver replete with LRPPRC. Pharmacological activators and inhibitors of the cellular respiration respectively increased and decreased the [NAD+]/[NADH] ratio, indicating respiration-mediated control of the [NAD+]/[NADH] ratio. Supporting a prominent role for NAD+, increasing the concentration of NAD+ stimulated complete oxidation of fatty acids. Importantly, NAD+ rescued impaired fatty acid oxidation in hepatocytes deficient for either OXPHOS or SIRT3. These data are consistent with a model whereby augmented hepatic OXPHOS increases NAD+, which in turn promotes complete oxidation of fatty acids and protects against NAFLD.

Ectopic vesicular glutamate release at the optic nerve head and axon loss in mouse experimental glaucoma.

PubMed

Fu, Christine T; Sretavan, David W

2012-11-07

Although clinical and experimental observations indicate that the optic nerve head (ONH) is a major site of axon degeneration in glaucoma, the mechanisms by which local retinal ganglion cell (RGC) axons are injured and damage spreads among axons remain poorly defined. Using a laser-induced ocular hypertension (LIOH) mouse model of glaucoma, we found that within 48 h of intraocular pressure elevation, RGC axon segments within the ONH exhibited ectopic accumulation and colocalization of multiple components of the glutamatergic presynaptic machinery including the vesicular glutamate transporter VGLUT2, several synaptic vesicle marker proteins, glutamate, the soluble N-ethylmaleimide-sensitive factor attachment protein receptor complex and active zone cytomatrix components, as well as ultrastructurally identified, synaptophysin-containing vesicles. Ectopic vesicle exocytosis and glutamate release were detected in acute preparations of the LIOH ONH. Immunolocalization and analysis using the ionotropic receptor channel-permeant cation agmatine indicated that ONH axon segments and glia expressed glutamate receptors, and these receptors were more active after LIOH compared with controls. Pharmacological antagonism of glutamate receptors and neuronal activity resulted in increased RGC axon sparing in vivo. Furthermore, in vivo RGC-specific genetic disruption of the vesicular glutamate transporter VGLUT2 or the obligatory NMDA receptor subunit NR1 promoted axon survival in experimental glaucoma. As the inhibition of ectopic glutamate vesicular release or glutamate receptivity can independently modify the severity of RGC axon loss, synaptic release mechanisms may provide useful therapeutic entry points into glaucomatous axon degeneration.

LAMP-2C Inhibits MHC Class II Presentation of Cytoplasmic Antigens by Disrupting Chaperone-Mediated Autophagy.

PubMed

Pérez, Liliana; McLetchie, Shawna; Gardiner, Gail J; Deffit, Sarah N; Zhou, Delu; Blum, Janice S

2016-03-15

Cells use multiple autophagy pathways to sequester macromolecules, senescent organelles, and pathogens. Several conserved isoforms of the lysosome-associated membrane protein-2 (LAMP-2) regulate these pathways influencing immune recognition and responses. LAMP-2A is required for chaperone-mediated autophagy (CMA), which promotes Ag capture and MHC class II (MHCII) presentation in B cells and signaling in T cells. LAMP-2B regulates lysosome maturation to impact macroautophagy and phagocytosis. Yet, far less is known about LAMP-2C function. Whereas LAMP2A and LAMP2B mRNA were broadly detected in human tissues, LAMP2C expression was more limited. Transcripts for the three LAMP2 isoforms increased with B cell activation, although specific gene induction varied depending on TLR versus BCR engagement. To examine LAMP-2C function in human B cells and specifically its role in Ag presentation, we used ectopic gene expression. Increased LAMP-2C expression in B cells did not alter MHCII expression or invariant chain processing, but did perturb cytoplasmic Ag presentation via CMA. MHCII presentation of epitopes from exogenous and membrane Ags was not affected by LAMP-2C expression in B cells. Similarly, changes in B cell LAMP-2C expression did not impact macroautophagy. The gene expression of other LAMP2 isoforms and proteasome and lysosomal proteases activities were unperturbed by LAMP-2C ectopic expression. LAMP-2C levels modulated the steady-state expression of several cytoplasmic proteins that are targeted for degradation by CMA and diminished peptide translocation via this pathway. Thus, LAMP-2C serves as a natural inhibitor of CMA that can selectively skew MHCII presentation of cytoplasmic Ags. Copyright © 2016 by The American Association of Immunologists, Inc.

LAMP-2C inhibits MHC class II presentation of cytoplasmic antigens by disrupting chaperone-mediated autophagy

PubMed Central

Pérez, Liliana; McLetchie, Shawna; Gardiner, Gail J.; Deffit, Sarah N.; Zhou, Delu; Blum, Janice S.

2016-01-01

Cells utilize multiple autophagy pathways to sequester macromolecules, senescent organelles, and pathogens. Several conserved isoforms of the lysosome-associated membrane protein (LAMP)-2 regulate these pathways influencing immune recognition and responses. LAMP-2A is required for chaperone-mediated autophagy (CMA) which promotes Ag capture and MHC class II (MHCII) presentation in B cells and signaling in T cells. LAMP-2B regulates lysosome maturation to impact macroautophagy (MA) and phagocytosis. Yet, far less is known about LAMP-2C function. While LAMP2A and LAMP2B mRNA were broadly detected in human tissues, LAMP2C expression was more limited. Transcripts for the three LAMP2 isoforms increased with B cell activation, although specific gene induction varied depending on TLR versus BCR engagement. To examine LAMP-2C function in human B cells and specifically its role in Ag presentation, ectopic gene expression was used. Increased LAMP-2C expression in B cells did not alter MHCII expression or invariant chain processing, but did perturb cytoplasmic Ag presentation via CMA. MHCII presentation of epitopes from exogenous and membrane Ags was not affected by LAMP-2C expression in B cells. Similarly, changes in B cell LAMP-2C expression did not impact MA. The gene expression of other LAMP2 isoforms as well as the proteasome and lysosomal proteases activities were unperturbed by LAMP-2C ectopic expression. LAMP-2C levels modulated the steady-state expression of several cytoplasmic proteins which are targeted for degradation by CMA and diminished peptide translocation via this pathway. Thus, LAMP-2C serves as a natural inhibitor of CMA which can selectively skew MHCII presentation of cytoplasmic Ags. PMID:26856698

Argonaute2 and LaminB modulate gene expression by controlling chromatin topology

PubMed Central

Nazer, Ezequiel; Dale, Ryan K.; Chinen, Madoka; Radmanesh, Behram

2018-01-01

Drosophila Argonaute2 (AGO2) has been shown to regulate expression of certain loci in an RNA interference (RNAi)-independent manner, but its genome-wide function on chromatin remains unknown. Here, we identified the nuclear scaffolding protein LaminB as a novel interactor of AGO2. When either AGO2 or LaminB are depleted in Kc cells, similar transcription changes are observed genome-wide. In particular, changes in expression occur mainly in active or potentially active chromatin, both inside and outside LaminB-associated domains (LADs). Furthermore, we identified a somatic target of AGO2 transcriptional repression, no hitter (nht), which is immersed in a LAD located within a repressive topologically-associated domain (TAD). Null mutation but not catalytic inactivation of AGO2 leads to ectopic expression of nht and downstream spermatogenesis genes. Depletion of either AGO2 or LaminB results in reduced looping interactions within the nht TAD as well as ectopic inter-TAD interactions, as detected by 4C-seq analysis. Overall, our findings reveal coordination of AGO2 and LaminB function to dictate genome architecture and thereby regulate gene expression. PMID:29529026

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhong, Yiming; Program in Molecular, Cellular, and Developmental Biology, The Ohio State University, Columbus, OH; Sullenbarger, Brent

Research highlights: {yields} HoxB4 overexpression in human TF1 cells increased the expression of CD61 and CD41a. {yields} HoxB4 fusion protein enhanced megakaryocytic development of CD34{sup +} cord blood cells. {yields} Ectopic HoxB4 increased Tpo receptor expression and decreased c-Myb expression. {yields} HoxB4 RNA silencing increased c-Myb expression and decreased Fli-1 expression. -- Abstract: In order to produce clinically useful quantities of platelets ex vivo we may need to firstly enhance early self-renewal of hematopoietic stem cells (HSCs) and/or megakaryocyte (Mk) progenitors. The homeodomain transcription factor HoxB4 has been shown to be an important regulator of stem cell renewal and hematopoiesis;more » however, its effect on megakaryopoiesis is unclear. In this study, we investigated the effect of HoxB4 overexpression or RNA silencing on megakaryocytic development in the human TF1 progenitor cell line; we then used recombinant tPTD-HoxB4 fusion protein to study the effect of exogenous HoxB4 on megakaryocytic development of human CD34 positively-selected cord blood cells. We found that ectopic HoxB4 in TF1 cells increased the antigen expression of CD61and CD41a, increased the gene expression of thrombopoietin receptor (TpoR), Scl-1, Cyclin D1, Fog-1 and Fli-1 while it decreased c-Myb expression. HoxB4 RNA silencing in TF1 cells decreased the expression of CD61 and CD41a and decreased Fli-1 expression while it increased the expression of c-Myb. Recombinant tPTD-HoxB4 fusion protein increased the percentages and absolute numbers of CD41a and CD61 positive cells during megakaryocytic differentiation of CD34 positively-selected cord blood cells and increased the numbers of colony-forming unit-megakaryocyte (CFU-Mk). Adding tPTD-HoxB4 fusion protein increased the gene expression of TpoR, Cyclin D1, Fog-1 and Fli-1 while it inhibited c-Myb expression. Our data suggest that increased HoxB4 enhanced early megakaryocytic development in human TF1 cells and CD34 positively-selected cord blood cells primarily by upregulating TpoR and Fli-1 expression and downregulating c-Myb expression. Increasing HoxB4 expression or adding recombinant HoxB4 protein might be a way to expand Mks for the production of platelets for use in transfusion medicine.« less

The Metastasis Efficiency Modifier Ribosomal RNA Processing 1 Homolog B (RRP1B) Is a Chromatin-associated Factor*

PubMed Central

Crawford, Nigel P. S.; Yang, Hailiu; Mattaini, Katherine R.; Hunter, Kent W.

2009-01-01

There is accumulating evidence for a role of germ line variation in breast cancer metastasis. We have recently identified a novel metastasis susceptibility gene, Rrp1b (ribosomal RNA processing 1 homolog B). Overexpression of Rrp1b in a mouse mammary tumor cell line induces a gene expression signature that predicts survival in breast cancer. Here we extend the analysis of RRP1B function by demonstrating that the Rrp1b activation gene expression signature accurately predicted the outcome in three of four publicly available breast carcinoma gene expression data sets. In addition, we provide insights into the mechanism of RRP1B. Tandem affinity purification demonstrated that RRP1B physically interacts with many nucleosome binding factors, including histone H1X, poly(ADP-ribose) polymerase 1, TRIM28 (tripartite motif-containing 28), and CSDA (cold shock domain protein A). Co-immunofluorescence and co-immunoprecipitation confirmed these interactions and also interactions with heterochromatin protein-1α and acetyl-histone H4 lysine 5. Finally, we investigated the effects of ectopic expression of an RRP1B allelic variant previously associated with improved survival in breast cancer. Gene expression analyses demonstrate that, compared with ectopic expression of wild type RRP1B in HeLa cells, the variant RRP1B differentially modulates various transcription factors controlled by TRIM28 and CSDA. These data suggest that RRP1B, a tumor progression and metastasis susceptibility candidate gene, is potentially a dynamic modulator of transcription and chromatin structure. PMID:19710015

Characterization of a citrus R2R3-MYB transcription factor that regulates the flavonol and hydroxycinnamic acid biosynthesis

USDA-ARS?s Scientific Manuscript database

Flavonols and hydroxycinnamic acids are important phenylpropanoid metabolites in plants. In this study, we isolated and characterized a citrus R2R3-MYB transcription factor CsMYBF1, encoding a protein belonging to the flavonol-specific MYB subgroup. Ectopic expression of CsMYBF1 in tomato led to an ...

Ectopic expression of Arabidopsis genes encoding salicylic acid- and jasmonic acid-related proteins confers partial resistance to soybean cyst nematode (Heterodera glycines) in transgenic soybean roots

USDA-ARS?s Scientific Manuscript database

Background. Extensive studies using the model system Arabidopsis thaliana to elucidate plant defense signaling and pathway networks indicate that salicylic acid (SA) is the key hormone triggering the plant defense response against biotrophic and hemi-biotrophic pathogens, while jasmonic acid (JA) an...

Quantitative proteomic analysis of human lung tumor xenografts treated with the ectopic ATP synthase inhibitor citreoviridin.

PubMed

Wu, Yi-Hsuan; Hu, Chia-Wei; Chien, Chih-Wei; Chen, Yu-Ju; Huang, Hsuan-Cheng; Juan, Hsueh-Fen

2013-01-01

ATP synthase is present on the plasma membrane of several types of cancer cells. Citreoviridin, an ATP synthase inhibitor, selectively suppresses the proliferation and growth of lung cancer without affecting normal cells. However, the global effects of targeting ectopic ATP synthase in vivo have not been well defined. In this study, we performed quantitative proteomic analysis using isobaric tags for relative and absolute quantitation (iTRAQ) and provided a comprehensive insight into the complicated regulation by citreoviridin in a lung cancer xenograft model. With high reproducibility of the quantitation, we obtained quantitative proteomic profiling with 2,659 proteins identified. Bioinformatics analysis of the 141 differentially expressed proteins selected by their relative abundance revealed that citreoviridin induces alterations in the expression of glucose metabolism-related enzymes in lung cancer. The up-regulation of enzymes involved in gluconeogenesis and storage of glucose indicated that citreoviridin may reduce the glycolytic intermediates for macromolecule synthesis and inhibit cell proliferation. Using comprehensive proteomics, the results identify metabolic aspects that help explain the antitumorigenic effect of citreoviridin in lung cancer, which may lead to a better understanding of the links between metabolism and tumorigenesis in cancer therapy.

Enhanced growth of influenza A virus by coinfection with human parainfluenza virus type 2.

PubMed

Goto, Hideo; Ihira, Hironobu; Morishita, Keiichi; Tsuchiya, Mitsuki; Ohta, Keisuke; Yumine, Natsuko; Tsurudome, Masato; Nishio, Machiko

2016-06-01

It has been reported that dual or multiple viruses can coinfect epithelial cells of the respiratory tract. However, little has been reported on in vitro interactions of coinfected viruses. To explore how coinfection of different viruses affects their biological property, we examined growth of influenza A virus (IAV) and human parainfluenza virus type 2 (hPIV2) during coinfection of Vero cells. We found that IAV growth was enhanced by coinfection with hPIV2. The enhanced growth of IAV was not reproduced by coinfection with an hPIV2 mutant with reduced cell fusion activity, or by ectopic expression of the V protein of hPIV2. In contrast, induction of cell fusion by ectopic expression of the hPIV2 HN and F proteins augments IAV growth. hPIV2 coinfection supported IAV growth in cells originated from the respiratory epithelium. The enhancement correlated closely with cell fusion ability of hPIV2 in those cells. These results indicate that cell fusion induced by hPIV2 infection is beneficial to IAV replication and that enhanced viral replication by coinfection with different viruses can modify their pathological consequences.

Quantitative Proteomic Analysis of Human Lung Tumor Xenografts Treated with the Ectopic ATP Synthase Inhibitor Citreoviridin

PubMed Central

Wu, Yi-Hsuan; Hu, Chia-Wei; Chien, Chih-Wei; Chen, Yu-Ju; Huang, Hsuan-Cheng; Juan, Hsueh-Fen

2013-01-01

ATP synthase is present on the plasma membrane of several types of cancer cells. Citreoviridin, an ATP synthase inhibitor, selectively suppresses the proliferation and growth of lung cancer without affecting normal cells. However, the global effects of targeting ectopic ATP synthase in vivo have not been well defined. In this study, we performed quantitative proteomic analysis using isobaric tags for relative and absolute quantitation (iTRAQ) and provided a comprehensive insight into the complicated regulation by citreoviridin in a lung cancer xenograft model. With high reproducibility of the quantitation, we obtained quantitative proteomic profiling with 2,659 proteins identified. Bioinformatics analysis of the 141 differentially expressed proteins selected by their relative abundance revealed that citreoviridin induces alterations in the expression of glucose metabolism-related enzymes in lung cancer. The up-regulation of enzymes involved in gluconeogenesis and storage of glucose indicated that citreoviridin may reduce the glycolytic intermediates for macromolecule synthesis and inhibit cell proliferation. Using comprehensive proteomics, the results identify metabolic aspects that help explain the antitumorigenic effect of citreoviridin in lung cancer, which may lead to a better understanding of the links between metabolism and tumorigenesis in cancer therapy. PMID:23990911

p38 phosphorylation in medullary microglia mediates ectopic orofacial inflammatory pain in rats.

PubMed

Kiyomoto, Masaaki; Shinoda, Masamichi; Honda, Kuniya; Nakaya, Yuka; Dezawa, Ko; Katagiri, Ayano; Kamakura, Satoshi; Inoue, Tomio; Iwata, Koichi

2015-08-12

Orofacial inflammatory pain is likely to accompany referred pain in uninflamed orofacial structures. The ectopic pain precludes precise diagnosis and makes treatment problematic, because the underlying mechanism is not well understood. Using the established ectopic orofacial pain model induced by complete Freund's adjuvant (CFA) injection into trapezius muscle, we analyzed the possible role of p38 phosphorylation in activated microglia in ectopic orofacial pain. Mechanical allodynia in the lateral facial skin was induced following trapezius muscle inflammation, which accompanied microglial activation with p38 phosphorylation and hyperexcitability of wide dynamic range (WDR) neurons in the trigeminal spinal subnucleus caudalis (Vc). Intra-cisterna successive administration of a p38 mitogen-activated protein kinase selective inhibitor, SB203580, suppressed microglial activation and its phosphorylation of p38. Moreover, SB203580 administration completely suppressed mechanical allodynia in the lateral facial skin and enhanced WDR neuronal excitability in Vc. Microglial interleukin-1β over-expression in Vc was induced by trapezius muscle inflammation, which was significantly suppressed by SB203580 administration. These findings indicate that microglia, activated via p38 phosphorylation, play a pivotal role in WDR neuronal hyperexcitability, which accounts for the mechanical hypersensitivity in the lateral facial skin associated with trapezius muscle inflammation.

nlz gene family is required for hindbrain patterning in the zebrafish.

PubMed

Hoyle, Jacqueline; Tang, Yixin P; Wiellette, Elizabeth L; Wardle, Fiona C; Sive, Hazel

2004-04-01

This study describes the conserved nlz gene family whose members encode unusual zinc finger proteins. In the zebrafish neurectoderm, both nlz1 and the newly isolated nlz2 are expressed in the presumptive hindbrain and midbrain/hindbrain boundary, where expression of nlz1 is dependent on pax2a. In addition, nlz2 is uniquely expressed more anteriorly, in the presumptive midbrain and diencephalon. Overexpression of Nlz proteins during gastrula stages inhibits hindbrain development. In particular, ectopically expressed Nlz1 inhibits formation of future rhombomeres 2 and 3 (r2, r3), whereas neighboring r1 and r4 are not affected. Conversely, simultaneous reduction of Nlz1 and Nlz2 protein function by expression of antisense morpholino-modified oligomers leads to expansion of future r3 and r5, with associated loss of r4. These data indicate that one function of the nlz gene family is to specify or maintain r4 identity, and to limit r3 and r5 during hindbrain formation. Copyright 2004 Wiley-Liss, Inc.

Down-regulation of Transducin-Like Enhancer of Split protein 4 in hepatocellular carcinoma promotes cell proliferation and epithelial-Mesenchymal-Transition

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wu, Xiao-cai; Xiao, Cui-cui; Li, Hua

Background: Transducin-Like Enhancer of Split protein 4 (TLE4) has been reported to be involved in some subsets of acute myeloid leukemia and colorectal cancer. In the present study, we aimed to explore the role of TLE4 in tumorigenesis and cancer progression in hepatocellular carcinoma (HCC). Methods: The expression pattern of TLE4 in HCC was determined by Western-blot and qRT-PCR, gain-of-function and loss-of-function was used to explore the biological role of TLE4 in HCC cells. A xenograft model was established to confirm its effects on proliferation. Results: The protein expression levels of TLE4 were significantly down-regulated in HCC tissues compared tomore » matched adjacent normal liver tissues. In vitro, down-regulation of TLE4 in Huh7 or SMMC-7721 promoted cell proliferation and ectopical expression of TLE4 in Hep3B or Bel-7404 suppressed cell proliferation. In addition, the cell colony formation ability was enhanced after down-regulation of TLE4 expression in Huh-7 but suppressed after over-expression in Hep3B. Furthermore, down-regulation of TLE4 increased the cell invasion ability, as well as increased the expression level of Vimentin and decreased that of E-cadherin, indicating a phenotype of epithelial-mesenchymal transition (EMT) in HCC cells. On the contrary, ectopical expression of TLE4 in HCC cells decreased the cell invasion ability and inhibited EMT. In vivo, compared to control group, xenograft tumor volumes were significantly decreased in TLE4 overexpression group. Conclusions: These results demonstrated that TLE4 might play important regulatory roles in cellular proliferation and EMT process in HCC. - Highlights: • TLE4 is significantly down-regulated in HCC samples. • Down regulated of TLE4 in HCC cells promotes cell proliferation. • Down regulated of TLE4 in HCC cells promotes epithelial-to-mesenchymal transition.« less

A soybean MADS-box protein modulates floral organ numbers, petal identity and sterility

PubMed Central

2014-01-01

Background The MADS-box transcription factors play fundamental roles in reproductive developmental control. Although the roles of many plant MADS-box proteins have been extensively studied, there are almost no functional studies of them in soybean, an important protein and oil crop in the world. In addition, the MADS-box protein orthologs may have species-specific functions. Controlling male fertility is an important goal in plant hybrid breeding but is difficult in some crops like soybean. The morphological structure of soybean flowers prevents the cross-pollination. Understanding the molecular mechanisms for floral development will aid in engineering new sterile materials that could be applied in hybrid breeding programs in soybean. Result Through microarray analysis, a flower-enriched gene in soybean was selected and designated as GmMADS28. GmMADS28 belongs to AGL9/SEP subfamily of MADS-box proteins, localized in nucleus and showed specific expression patterns in floral meristems as well as stamen and petal primordia. Expression of GmMADS28 in the stamens and petals of a soybean mutant NJS-10Hfs whose stamens are converted into petals was higher than in those of wild-type plants. Constitutive expression of GmMADS28 in tobacco promoted early flowering and converted stamens and sepals to petals. Interestingly, transgenic plants increased the numbers of sepal, petal and stamen from five to six and exhibited male sterility due to the shortened and curly filaments and the failure of pollen release from the anthers. The ectopic expression of GmMADS28 was found to be sufficient to activate expression of tobacco homologs of SOC1, LEAFY, AGL8/FUL, and DEF. In addition, we observed the interactions of GmMADS28 with soybean homologs of SOC1, AP1, and AGL8/FUL proteins. Conclusion In this study, we observed the roles of GmMADS28 in the regulation of floral organ number and petal identity. Compared to other plant AGL9/SEP proteins, GmMADS28 specifically regulates floral organ number, filament length and pollen release. The sterility caused by the ectopic expression of GmMADS28 offers a promising way to genetically produce new sterile material that could potentially be applied in the hybrid breeding of crops like soybean. PMID:24693922

A soybean MADS-box protein modulates floral organ numbers, petal identity and sterility.

PubMed

Huang, Fang; Xu, Guangli; Chi, Yingjun; Liu, Haicui; Xue, Qian; Zhao, Tuanjie; Gai, Junyi; Yu, Deyue

2014-04-02

The MADS-box transcription factors play fundamental roles in reproductive developmental control. Although the roles of many plant MADS-box proteins have been extensively studied, there are almost no functional studies of them in soybean, an important protein and oil crop in the world. In addition, the MADS-box protein orthologs may have species-specific functions. Controlling male fertility is an important goal in plant hybrid breeding but is difficult in some crops like soybean. The morphological structure of soybean flowers prevents the cross-pollination. Understanding the molecular mechanisms for floral development will aid in engineering new sterile materials that could be applied in hybrid breeding programs in soybean. Through microarray analysis, a flower-enriched gene in soybean was selected and designated as GmMADS28. GmMADS28 belongs to AGL9/SEP subfamily of MADS-box proteins, localized in nucleus and showed specific expression patterns in floral meristems as well as stamen and petal primordia. Expression of GmMADS28 in the stamens and petals of a soybean mutant NJS-10Hfs whose stamens are converted into petals was higher than in those of wild-type plants. Constitutive expression of GmMADS28 in tobacco promoted early flowering and converted stamens and sepals to petals. Interestingly, transgenic plants increased the numbers of sepal, petal and stamen from five to six and exhibited male sterility due to the shortened and curly filaments and the failure of pollen release from the anthers. The ectopic expression of GmMADS28 was found to be sufficient to activate expression of tobacco homologs of SOC1, LEAFY, AGL8/FUL, and DEF. In addition, we observed the interactions of GmMADS28 with soybean homologs of SOC1, AP1, and AGL8/FUL proteins. In this study, we observed the roles of GmMADS28 in the regulation of floral organ number and petal identity. Compared to other plant AGL9/SEP proteins, GmMADS28 specifically regulates floral organ number, filament length and pollen release. The sterility caused by the ectopic expression of GmMADS28 offers a promising way to genetically produce new sterile material that could potentially be applied in the hybrid breeding of crops like soybean.

Interferon regulatory factor-1 binds c-Cbl, enhances mitogen activated protein kinase signaling and promotes retinoic acid-induced differentiation of HL-60 human myelo-monoblastic leukemia cells.

PubMed

Shen, Miaoqing; Bunaciu, Rodica P; Congleton, Johanna; Jensen, Holly A; Sayam, Lavanya G; Varner, Jeffrey D; Yen, Andrew

2011-12-01

All-trans retinoic acid (RA) and interferons (IFNs) have efficacy in treating certain leukemias and lymphomas, respectively, motivating interest in their mechanism of action to improve therapy. Both RA and IFNs induce interferon regulatory factor-1 (IRF-1). We find that in HL-60 myeloblastic leukemia cells which undergo mitogen activated protien kinase (MAPK)-dependent myeloid differentiation in response to RA, IRF-1 propels differentiation. RA induces MAPK-dependent expression of IRF-1. IRF-1 binds c-Cbl, a MAPK related adaptor. Ectopic IRF-1 expression causes CD38 expression and activation of the Raf/MEK/ERK axis, and enhances RA-induced differentiation by augmenting CD38, CD11b, respiratory burst and G0 arrest. Ectopic IRF-1 expression also decreases the activity of aldehyde dehydrogenase 1, a stem cell marker, and enhances RA-induced ALDH1 down-regulation. Interestingly, expression of aryl hydrocarbon receptor (AhR), which is RA-induced and known to down-regulate Oct4 and drive RA-induced differentiation, also enhances IRF-1 expression. The data are consistent with a model whereby IRF-1 acts downstream of RA and AhR to enhance Raf/MEK/ERK activation and propel differentiation.

Ectopic expression of SUPERMAN suppresses development of petals and stamens.

PubMed

Yun, Jae-Young; Weigel, Detlef; Lee, Ilha

2002-01-01

The floral regulatory gene SUPERMAN (SUP) encodes a C2H2 type zinc finger protein that is required for maintaining boundaries between floral organs in Arabidopsis. It has been proposed that the main function of SUP is to balance cell proliferation in the third and fourth whorl of developing flowers, thereby maintaining the boundaries between the two whorls. To gain further insight into the function of SUP, we have ectopically expressed SUP using the promoter of APETALA1 (AP1), a gene that is initially expressed throughout floral meristems and later becomes restricted to the first and second whorls. Flowers of AP1::SUP plants have fewer floral organs, consistent with an effect of SUP on cell proliferation. In addition, the AP1::SUP transgene caused the conversion of petals to sepals and suppressed the development of stamens. The expression of the B function homeotic gene APETALA3 (AP3) and its regulator UNUSUAL FLORAL ORGANS (UFO) were delayed and reduced in AP1::SUP flowers. However, SUP does not act merely through UFO, as constitutive expression of UFO did not rescue the defects in petal and stamen development in AP1::SUP flowers. Together, these results suggest that SUP has both indirect and direct effects on the expression of B function homeotic genes.

Ectopic Expression of Pumpkin NAC Transcription Factor CmNAC1 Improves Multiple Abiotic Stress Tolerance in Arabidopsis

PubMed Central

Cao, Haishun; Wang, Li; Nawaz, Muhammad A.; Niu, Mengliang; Sun, Jingyu; Xie, Junjun; Kong, Qiusheng; Huang, Yuan; Cheng, Fei; Bie, Zhilong

2017-01-01

Drought, cold and salinity are the major environmental stresses that limit agricultural productivity. NAC transcription factors regulate the stress response in plants. Pumpkin (Cucurbita moschata) is an important cucurbit vegetable crop and it has strong resistance to abiotic stress; however, the biological functions of stress-related NAC genes in this crop are largely unknown. This study reports the function of CmNAC1, a stress-responsive pumpkin NAC domain protein. The CmNAC1-GFP fusion protein was transiently expressed in tobacco leaves for subcellular localization analysis, and we found that CmNAC1 is localized in the nucleus. Transactivation assay in yeast cells revealed that CmNAC1 functions as a transcription activator, and its transactivation domain is located in the C-terminus. CmNAC1 was ubiquitously expressed in different organs, and its transcript was induced by salinity, cold, dehydration, H2O2, and abscisic acid (ABA) treatment. Furthermore, the ectopic expression (EE) of CmNAC1 in Arabidopsis led to ABA hypersensitivity and enhanced tolerance to salinity, drought and cold stress. In addition, five ABA-responsive elements were enriched in CmNAC1 promoter. The CmNAC1-EE plants exhibited different root architecture, leaf morphology, and significantly high concentration of ABA compared with WT Arabidopsis under normal conditions. Our results indicated that CmNAC1 is a critical factor in ABA signaling pathways and it can be utilized in transgenic breeding to improve the abiotic stress tolerance of crops. PMID:29234347

An efficient transgenic system by TA cloning vectors and RNAi for C. elegans

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gengyo-Ando, Keiko; CREST, JST, 4-1-8 Hon-cho, Kawaguchi, Saitama 332-0012; Yoshina, Sawako

2006-11-03

In the nematode, transgenic analyses have been performed by microinjection of DNA from various sources into the syncytium gonad. To expedite these transgenic analyses, we solved two potential problems in this work. First, we constructed an efficient TA-cloning vector system which is useful for any promoter. By amplifying the genomic DNA fragments which contain regulatory sequences with or without the coding region, we could easily construct plasmids expressing fluorescent protein fusion without considering restriction sites. We could dissect motor neurons with three colors in a single animal. Second, we used feeding RNAi to isolate transgenic strains which express lag-2::venus fusionmore » gene. We found that the fusion protein is toxic when ectopically expressed in embryos but is functional to rescue a loss of function mutant in the lag-2 gene. Thus, the transgenic system described here should be useful to examine the protein function in the nematode.« less

Glucose Oxidase Induces Cellular Senescence in Immortal Renal Cells through ILK by Downregulating Klotho Gene Expression

PubMed Central

Troyano-Suárez, Nuria; del Nogal-Avila, María; Mora, Inés; Sosa, Patricia; López-Ongil, Susana; Rodriguez-Puyol, Diego; Olmos, Gemma; Ruíz-Torres, María Piedad

2015-01-01

Cellular senescence can be prematurely induced by oxidative stress involved in aging. In this work, we were searching for novel intermediaries in oxidative stress-induced senescence, focusing our interest on integrin-linked kinase (ILK), a scaffold protein at cell-extracellular matrix (ECM) adhesion sites, and on the Klotho gene. Cultured renal cells were treated with glucose oxidase (GOx) for long time periods. GOx induced senescence, increasing senescence associated β-galactosidase activity and the expression of p16. In parallel, GOx increased ILK protein expression and activity. Ectopic overexpression of ILK in cells increased p16 expression, even in the absence of GOx, whereas downregulation of ILK inhibited the increase in p16 due to oxidative stress. Additionally, GOx reduced Klotho gene expression and cells overexpressing Klotho protein did not undergo senescence after GOx addition. We demonstrated a direct link between ILK and Klotho since silencing ILK expression in cells and mice increases Klotho expression and reduces p53 and p16 expression in renal cortex. In conclusion, oxidative stress induces cellular senescence in kidney cells by increasing ILK protein expression and activity, which in turn reduces Klotho expression. We hereby present ILK as a novel downregulator of Klotho gene expression. PMID:26583057

Glucose Oxidase Induces Cellular Senescence in Immortal Renal Cells through ILK by Downregulating Klotho Gene Expression.

PubMed

Troyano-Suárez, Nuria; del Nogal-Avila, María; Mora, Inés; Sosa, Patricia; López-Ongil, Susana; Rodriguez-Puyol, Diego; Olmos, Gemma; Ruíz-Torres, María Piedad

2015-01-01

Cellular senescence can be prematurely induced by oxidative stress involved in aging. In this work, we were searching for novel intermediaries in oxidative stress-induced senescence, focusing our interest on integrin-linked kinase (ILK), a scaffold protein at cell-extracellular matrix (ECM) adhesion sites, and on the Klotho gene. Cultured renal cells were treated with glucose oxidase (GOx) for long time periods. GOx induced senescence, increasing senescence associated β-galactosidase activity and the expression of p16. In parallel, GOx increased ILK protein expression and activity. Ectopic overexpression of ILK in cells increased p16 expression, even in the absence of GOx, whereas downregulation of ILK inhibited the increase in p16 due to oxidative stress. Additionally, GOx reduced Klotho gene expression and cells overexpressing Klotho protein did not undergo senescence after GOx addition. We demonstrated a direct link between ILK and Klotho since silencing ILK expression in cells and mice increases Klotho expression and reduces p53 and p16 expression in renal cortex. In conclusion, oxidative stress induces cellular senescence in kidney cells by increasing ILK protein expression and activity, which in turn reduces Klotho expression. We hereby present ILK as a novel downregulator of Klotho gene expression.

Arabidopsis and tobacco plants ectopically expressing the soybean antiquitin-like ALDH7 gene display enhanced tolerance to drought, salinity, and oxidative stress.

PubMed

Rodrigues, Simone M; Andrade, Maxuel O; Gomes, Ana Paula Soares; Damatta, Fabio M; Baracat-Pereira, Maria C; Fontes, Elizabeth P B

2006-01-01

Despite extensive studies in eukaryotic aldehyde dehydrogenases, functional information about the ALDH7 antiquitin-like proteins is lacking. A soybean antiquitin homologue gene, designated GmTP55, has been isolated which encodes a dehydrogenase motif-containing 55 kDa protein induced by dehydration and salt stress. GmTP55 is closely related to the stress-induced plant antiquitin-like proteins that belong to the ALDH7 family. Transgenic tobacco (Nicotiana tabacum) and Arabidopsis (Arabidopsis thaliana) plants constitutively expressing GmTP55 have been obtained in order to examine the physiological role of this enzyme under a variety of stress conditions. Ectopic expression of GmTP55 in both Arabidopsis and tobacco conferred tolerance to salinity during germination and to water deficit during plant growth. Under salt stress, the germination efficiency of both transgenic tobacco and Arabidopsis seeds was significantly higher than that of their control counterparts. Likewise, under progressive drought, the transgenic tobacco lines apparently kept the shoot turgidity to a normal level, which contrasted with the leaf wilt phenotype of control plants. The transgenic plants also exhibited an enhanced tolerance to H(2)O(2)- and paraquat-induced oxidative stress. Both GmTP55-expressing Arabidopsis and tobacco seeds germinated efficiently in medium supplemented with H(2)O(2), whereas the germination of control seeds was drastically impaired. Similarly, transgenic tobacco leaf discs treated with paraquat displayed a significant reduction in the necrotic lesions as compared with control leaves. These transgenic lines also exhibited a lower concentration of lipid peroxidation-derived reactive aldehydes under oxidative stress. These results suggest that antiquitin may be involved in adaptive responses mediated by a physiologically relevant detoxification pathway in plants.

Expression of GRIM-19 in adenomyosis and its possible role in pathogenesis.

PubMed

Wang, Jing; Deng, Xiaohui; Yang, Yang; Yang, Xingsheng; Kong, Beihua; Chao, Lan

2016-04-01

To study the expression of the gene associated with retinoid-interferon (IFN)-induced mortality 19 (GRIM-19) in the endometrial tissue of patients with adenomyosis and to describe the possible pathogenic mechanisms of this phenomenon. Experimental study using human samples and cell lines. University-affiliated hospital. Ectopic and eutopic endometrial tissues were obtained from 30 patients with adenomyosis, whereas normal endometrial specimens were obtained from 10 control patients without adenomyosis. Patients with rapid pathology report-confirmed adenomyosis were recruited, and eutopic and ectopic endometrial tissue samples were collected from patients who had undergone hysterectomies by either the transabdominal or laparoscopic method at Qilu Hospital. Normal endometrial tissue was collected from a group of control patients without adenomyosis. Immunohistochemistry (IHC) was performed to evaluate the expression of GRIM-19, phospho-signal transducer and activator of transcription 3 (Y705) (Y705) (pSTAT3(Y705)), and vascular endothelial growth factor (VEGF) in endometrial tissue samples. The protein levels of GRIM-19, pSTAT3(Y705), STAT3, and VEGF were detected by Western blot. Apoptosis in endometrial specimens was assayed by TUNEL. Immunohistochemistry with an antibody directed against CD34 was performed to detect new blood vessels in the endometrial tissue. GRIM-19 small interfering RNA and a recombinant plasmid carrying GRIM-19 were constructed to evaluate the effects of GRIM-19 on the downstream factors pSTAT3(Y705), STAT3, and VEGF in Ishikawa cells. The expression of GRIM-19 was down-regulated in the eutopic endometria of patients with adenomyosis compared with the endometria of patients in the control group, and it was further reduced in the endometrial glandular epithelial cells of adenomyotic lesions. Apoptosis was reduced in the eutopic endometrium compared with the control group, and it was significantly reduced in ectopic endometrial tissues. In addition, the ectopic and eutopic endometria of patients with adenomyosis displayed a much higher microvessel density. In the eutopic and ectopic endometria of patients with adenomyosis, the expression levels of pSTAT3(Y705) and VEGF were significantly higher than in the controls. Furthermore, down-regulation of GRIM-19 in Ishikawa cells significantly promoted the activation of both pSTAT3(Y705) and its dependent gene VEGF. Aberrant expression of GRIM-19 may be associated with adenomyosis through the regulation of apoptosis and angiogenesis. Copyright © 2016 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

E2F1-mediated human POMC expression in ectopic Cushing's syndrome.

PubMed

Araki, Takako; Liu, Ning-Ai; Tone, Yukiko; Cuevas-Ramos, Daniel; Heltsley, Roy; Tone, Masahide; Melmed, Shlomo

2016-11-01

Cushing's syndrome is caused by excessive adrenocorticotropic hormone (ACTH) secretion derived from pituitary corticotroph tumors (Cushing disease) or from non-pituitary tumors (ectopic Cushing's syndrome). Hypercortisolemic features of ectopic Cushing's syndrome are severe, and no definitive treatment for paraneoplastic ACTH excess is available. We aimed to identify subcellular therapeutic targets by elucidating transcriptional regulation of the human ACTH precursor POMC (proopiomelanocortin) and ACTH production in non-pituitary tumor cells and in cell lines derived from patients with ectopic Cushing's syndrome. We show that ectopic hPOMC transcription proceeds independently of pituitary-specific Tpit/Pitx1 and demonstrate a novel E2F1-mediated transcriptional mechanism regulating hPOMC We identify an E2F1 cluster binding to the proximal hPOMC promoter region (-42 to +68), with DNA-binding activity determined by the phosphorylation at Ser-337. hPOMC mRNA expression in cancer cells was upregulated (up to 40-fold) by the co-expression of E2F1 and its heterodimer partner DP1. Direct and indirect inhibitors of E2F1 activity suppressed hPOMC gene expression and ACTH by modifying E2F1 DNA-binding activity in ectopic Cushing's cell lines and primary tumor cells, and also suppressed paraneoplastic ACTH and cortisol levels in xenografted mice. E2F1-mediated hPOMC transcription is a potential target for suppressing ACTH production in ectopic Cushing's syndrome. © 2016 Society for Endocrinology.

Developmental Role and Auxin Responsiveness of Class III Homeodomain Leucine Zipper Gene Family Members in Rice1[C][W][OA

PubMed Central

Itoh, Jun-Ichi; Hibara, Ken-Ichiro; Sato, Yutaka; Nagato, Yasuo

2008-01-01

Members of the Class III homeodomain leucine zipper (Class III HD-Zip) gene family are central regulators of crucial aspects of plant development. To better understand the roles of five Class III HD-Zip genes in rice (Oryza sativa) development, we investigated their expression patterns, ectopic expression phenotypes, and auxin responsiveness. Four genes, OSHB1 to OSHB4, were expressed in a localized domain of the shoot apical meristem (SAM), the adaxial cells of leaf primordia, the leaf margins, and the xylem tissue of vascular bundles. In contrast, expression of OSHB5 was observed only in phloem tissue. Plants ectopically expressing microRNA166-resistant versions of the OSHB3 gene exhibited severe defects, including the ectopic production of leaf margins, shoots, and radialized leaves. The treatment of seedlings with auxin quickly induced ectopic OSHB3 expression in the entire region of the SAM, but not in other tissues. Furthermore, this ectopic expression of OSHB3 was correlated with leaf initiation defects. Our findings suggest that rice Class III HD-Zip genes have conserved functions with their homologs in Arabidopsis (Arabidopsis thaliana), but have also acquired specific developmental roles in grasses or monocots. In addition, some Class III HD-Zip genes may regulate the leaf initiation process in the SAM in an auxin-dependent manner. PMID:18567825

Gene array analysis reveals a common Runx transcriptional program controlling cell adhesion and survival

PubMed Central

Wotton, Sandy; Terry, Anne; Kilbey, Anna; Jenkins, Alma; Herzyk, Pawel; Cameron, Ewan; Neil, James C.

2008-01-01

The Runx genes play divergent roles in development and cancer, where they can act either as oncogenes or tumour suppressors. We compared the effects of ectopic Runx expression in established fibroblasts, where all three genes produce an indistinguishable phenotype entailing epithelioid morphology and increased cell survival under stress conditions. Gene array analysis revealed a strongly overlapping transcriptional signature, with no examples of opposing regulation of the same target gene. A common set of 50 highly regulated genes was identified after further filtering on regulation by inducible RUNX1-ER. This set revealed a strong bias towards genes with annotated roles in cancer and development, and a preponderance of targets encoding extracellular or surface proteins, reflecting the marked effects of Runx on cell adhesion. Furthermore, in silico prediction of resistance to glucocorticoid growth inhibition was confirmed in fibroblasts and lymphoid cells expressing ectopic Runx. The effects of fibroblast expression of common RUNX1 fusion oncoproteins (RUNX1-ETO, TEL-RUNX1, CBFB-MYH11) were also tested. While two direct Runx activation target genes were repressed (Ncam1, Rgc32), the fusion proteins appeared to disrupt regulation of down-regulated targets (Cebpd, Id2, Rgs2) rather than impose constitutive repression. These results elucidate the oncogenic potential of the Runx family and reveal novel targets for therapeutic inhibition. PMID:18560354

Selenoprotein W enhances skeletal muscle differentiation by inhibiting TAZ binding to 14-3-3 protein.

PubMed

Jeon, Yeong Ha; Park, Yong Hwan; Lee, Jea Hwang; Hong, Jeong-Ho; Kim, Ick Young

2014-07-01

Selenoprotein W (SelW) is expressed in various tissues, particularly in skeletal muscle. We have previously reported that SelW is up-regulated during C2C12 skeletal muscle differentiation and inhibits binding of 14-3-3 to its target proteins. 14-3-3 reduces myogenic differentiation by inhibiting nuclear translocation of transcriptional co-activator with PDZ-binding motif (TAZ). Phosphorylation of TAZ at Ser89 is required for binding to 14-3-3, leading to cytoplasmic retention of TAZ and a delay in myogenic differentiation. Here, we show that myogenic differentiation was delayed in SelW-knockdown C2C12 cells. Down-regulation of SelW also increased TAZ binding to 14-3-3, which eventually resulted in decreasing translocation of TAZ to the nucleus. However, phosphorylation of TAZ at Ser89 was not affected. Although phosphorylation of TAZ at Ser89 was sustained by the phosphatase inhibitor okadaic acid, nuclear translocation of TAZ was increased by ectopic expression of SelW. This result was due to decreased binding of TAZ to 14-3-3. We also found that the interaction between TAZ and MyoD was increased by ectopic expression of SelW. Taken together, these findings strongly demonstrate that SelW enhances C2C12 cell differentiation by inhibiting TAZ binding to 14-3-3. Copyright © 2014 Elsevier B.V. All rights reserved.

The ectopic expression of Pax4 in the mouse pancreas converts progenitor cells into alpha and subsequently beta cells.

PubMed

Collombat, Patrick; Xu, Xiaobo; Ravassard, Philippe; Sosa-Pineda, Beatriz; Dussaud, Sébastien; Billestrup, Nils; Madsen, Ole D; Serup, Palle; Heimberg, Harry; Mansouri, Ahmed

2009-08-07

We have previously reported that the loss of Arx and/or Pax4 gene activity leads to a shift in the fate of the different endocrine cell subtypes in the mouse pancreas, without affecting the total endocrine cell numbers. Here, we conditionally and ectopically express Pax4 using different cell-specific promoters and demonstrate that Pax4 forces endocrine precursor cells, as well as mature alpha cells, to adopt a beta cell destiny. This results in a glucagon deficiency that provokes a compensatory and continuous glucagon+ cell neogenesis requiring the re-expression of the proendocrine gene Ngn3. However, the newly formed alpha cells fail to correct the hypoglucagonemia since they subsequently acquire a beta cell phenotype upon Pax4 ectopic expression. Notably, this cycle of neogenesis and redifferentiation caused by ectopic expression of Pax4 in alpha cells is capable of restoring a functional beta cell mass and curing diabetes in animals that have been chemically depleted of beta cells.

PAF53 is essential in mammalian cells: CRISPR/Cas9 fails to eliminate PAF53 expression.

PubMed

Rothblum, Lawrence I; Rothblum, Katrina; Chang, Eugenie

2017-05-15

When mammalian cells are nutrient and/or growth factor deprived, exposed to inhibitors of protein synthesis, stressed by heat shock or grown to confluence, rDNA transcription is essentially shut off. Various mechanisms are available to accomplish this downshift in ribosome biogenesis. Muramatsu's laboratory (Hanada et al., 1996) first demonstrated that mammalian PAF53 was essential for specific rDNA transcription and that PAF53 levels were regulated in response to growth factors. While S. cerevisae A49, the homologue of vertebrate PAF53, is not essential for viability (Liljelund et al., 1992), deletion of yA49 results in colonies that grow at 6% of the wild type rate at 25°C. Experiments described by Wang et al. (2015) identified PAF53 as a gene "essential for optimal proliferation". However, they did not discriminate genes essential for viability. Hence, in order to resolve this question, we designed a series of experiments to determine if PAF53 was essential for cell survival. We set out to delete the gene product from mammalian cells using CRISPR/CAS9 technology. Human 293 cells were transfected with lentiCRISPR v2 carrying genes for various sgRNA that targeted PAF53. In some experiments, the cells were cotransfected in parallel with plasmids encoding FLAG-tagged mouse PAF53. After treating the transfected cells with puromycin (to select for the lentiCRISPR backbone), cells were cloned and analyzed by western blots for PAF53 expression. Genomic DNA was amplified across the "CRISPRd" exon, cloned and sequenced to identify mutated PAF53 genes. We obtained cell lines in which the endogenous PAF53 gene was "knocked out" only when we rescued with FLAG-PAF53. DNA sequencing demonstrated that in the absence of ectopic PAF53 expression, cells demonstrated unique means of surviving; including recombination or the utilization of alternative reading frames. We never observed a clone in which one PAF53 gene is expressed, unless there was also ectopic expression In the absence of ectopic gene expression, the gene products of both endogenous genes were expressed, irrespective of whether they were partially mutant proteins or not. Copyright © 2016 Elsevier B.V. All rights reserved.

Expression of LIM kinase 1 is associated with reversible G1/S phase arrest, chromosomal instability and prostate cancer.

PubMed

Davila, Monica; Jhala, Darshana; Ghosh, Debashis; Grizzle, William E; Chakrabarti, Ratna

2007-06-08

LIM kinase 1 (LIMK1), a LIM domain containing serine/threonine kinase, modulates actin dynamics through inactivation of the actin depolymerizing protein cofilin. Recent studies have indicated an important role of LIMK1 in growth and invasion of prostate and breast cancer cells; however, the molecular mechanism whereby LIMK1 induces tumor progression is unknown. In this study, we investigated the effects of ectopic expression of LIMK1 on cellular morphology, cell cycle progression and expression profile of LIMK1 in prostate tumors. Ectopic expression of LIMK1 in benign prostatic hyperplasia cells (BPH), which naturally express low levels of LIMK1, resulted in appearance of abnormal mitotic spindles, multiple centrosomes and smaller chromosomal masses. Furthermore, a transient G1/S phase arrest and delayed G2/M progression was observed in BPH cells expressing LIMK1. When treated with chemotherapeutic agent Taxol, no metaphase arrest was noted in these cells. We have also noted increased nuclear staining of LIMK1 in tumors with higher Gleason Scores and incidence of metastasis. Our results show that increased expression of LIMK1 results in chromosomal abnormalities, aberrant cell cycle progression and alteration of normal cellular response to microtubule stabilizing agent Taxol; and that LIMK1 expression may be associated with cancerous phenotype of the prostate.

Integrated Phloem Sap mRNA and Protein Expression Analysis Reveals Phytoplasma-infection Responses in Mulberry.

PubMed

Gai, Yingping; Yuan, Shuo-Shuo; Liu, Zhao-Yang; Zhao, Huai-Ning; Liu, Qi; Qin, Yong-Li; Fang, Li-Jing; Ji, Xian-Ling

2018-05-30

To gain insight into the response of mulberry to phytoplasma-infection, the expression profiles of mRNAs and proteins in mulberry phloem sap were examined. A total of 955 unigenes and 136 proteins were found to be differentially expressed between the healthy and infected phloem sap. These differentially expressed mRNAs and proteins are involved in signalling, hormone metabolism, stress responses, etc. Interestingly, we found that both the mRNA and protein levels of the major latex protein-like 329 ( MuMLPL329 ) gene were increased in the infected phloem saps. Expression of the MuMLPL329 gene was induced by pathogen inoculation and was responsive to jasmonic acid. Ectopic expression of MuMLPL329 in Arabidopsis enhances transgenic plant resistance to Botrytis cinerea, Pseudomonas syringae pv tomato DC3000 ( Pst. DC3000 ) and phytoplasma. Further analysis revealed that MuMLPL329 can enhance the expression of some defense genes and might be involved in altering flavonoid content resulting in increased resistance of plants to pathogen infection. Finally, the roles of the differentially expressed mRNAs and proteins and the potential molecular mechanisms of their changes were discussed. It was likely that the phytoplasma-responsive mRNAs and proteins in the phloem saps were involved in multiple pathways of mulberry responses to phytoplasma-infection, and their changes may be partially responsible for some symptoms in the phytoplasma infected plants. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

The adaptor protein Cindr regulates JNK activity to maintain epithelial sheet integrity.

PubMed

Yasin, Hannah W R; van Rensburg, Samuel H; Feiler, Christina E; Johnson, Ruth I

2016-02-15

Epithelia are essential barrier tissues that must be appropriately maintained for their correct function. To achieve this a plethora of protein interactions regulate epithelial cell number, structure and adhesion, and differentiation. Here we show that Cindr (the Drosophila Cin85 and Cd2ap ortholog) is required to maintain epithelial integrity. Reducing Cindr triggered cell delamination and movement. Most delaminating cells died. These behaviors were consistent with JNK activation previously associated with loss of epithelial integrity in response to ectopic oncogene activity. We confirmed a novel interaction between Cindr and Drosophila JNK (dJNK), which when perturbed caused inappropriate JNK signaling. Genetically reducing JNK signaling activity suppressed the effects of reducing Cindr. Furthermore, ectopic JNK signaling phenocopied loss of Cindr and was partially rescued by concomitant cindr over-expression. Thus, correct Cindr-dJNK stoichiometry is essential to maintain epithelial integrity and disturbing this balance may contribute to the pathogenesis of disease states, including cancer. Copyright © 2016 Elsevier Inc. All rights reserved.

Genetic analysis of an overlapping functional requirement for L1- and NCAM-type proteins during sensory axon guidance in Drosophila.

PubMed

Kristiansen, Lars V; Velasquez, Emma; Romani, Susana; Baars, Sigrid; Berezin, Vladimir; Bock, Elisabeth; Hortsch, Michael; Garcia-Alonso, Luis

2005-01-01

L1- and NCAM-type cell adhesion molecules represent distinct protein families that function as specific receptors for different axon guidance cues. However, both L1 and NCAM proteins promote axonal growth by inducing neuronal tyrosine kinase activity and are coexpressed in subsets of axon tracts in arthropods and vertebrates. We have studied the functional requirements for the Drosophila L1- and NCAM-type proteins, Neuroglian (Nrg) and Fasciclin II (FasII), during postembryonic sensory axon guidance. The rescue of the Neuroglian loss-of-function (LOF) phenotype by transgenically expressed L1- and NCAM-type proteins demonstrates a functional interchangeability between these proteins in Drosophila photoreceptor pioneer axons, where both proteins are normally coexpressed. In contrast, the ectopic expression of Fasciclin II in mechanosensory neurons causes a strong enhancement of the axonal misguidance phenotype. Moreover, our findings demonstrate that this functionally redundant specificity to mediate axon guidance has been conserved in their vertebrate homologs, L1-CAM and NCAM.

Bcl-2 and caspase-3 are major regulators in Agaricus blazei-induced human leukemic U937 cell apoptosis through dephoshorylation of Akt.

PubMed

Jin, Cheng-Yun; Moon, Dong-Oh; Choi, Yung Hyun; Lee, Jae-Dong; Kim, Gi-Young

2007-08-01

Agaricus blazei is a medicinal mushroom that possesses antimetastatic, antitumor, antimutagenic, and immunostimulating effects. However, the molecular mechanisms involved in A. blazei-mediated apoptosis remain unclear. In the present study, to elucidate the role of the Bcl-2 in A. blazei-mediated apoptosis, U937 cells were transfected with either empty vector (U937/vec) or vector containing cDNA encoding full-length Bcl-2 (U937/Bcl-2). As compared with U937/vec, U937/Bcl-2 cells exhibited a 4-fold greater expression of Bcl-2. Treatment of U937/vec with 1.0-4.0 mg/ml of A. blazei extract (ABE) for 24 h resulted in a significant induction of morphologic features indicative of apoptosis. In contrast, U937/Bcl-2 exposed to the same ABE treatment only exhibited a slight induction of apoptotic features. ABE-induced apoptosis was accompanied by downregulation of antiapoptotic proteins such as X-linked inhibitor of apoptosis protein (XIAP), inhibitor of apoptosis protein (cIAP)-2 and Bcl-2, activation of caspase-3, and cleavage of poly(ADP-ribose)polymerase (PARP). Ectopic expression of Bcl-2 was associated with significantly induced expression of antiapoptotic proteins, such as cIAP-2 and Bcl-2, but not XIAP. Ectopic expression of Bcl-2 also reduced caspase-3 activation and PARP cleavage in ABE treated U937 cells. Furthermore, treatment with the caspase-3 inhibitor z-DEVD-fmk was sufficient to restore cell viability following ABE treatment. This increase in viability was ascribed to downregulation of caspase-3 and blockage of PARP and PLC-gamma cleavage. ABE also triggered the downregulation of Akt, and combined treatment with LY294002 (an inhibitor of Akt) significantly decreased cell viability. The results indicated that major regulators of ABE-induced apoptosis in human leukemic U937 cells are Bcl-2 and caspase-3, which are associated with dephosphorylation of the Akt signal pathway.

Ectopic expression of Cripto-1 in transgenic mouse embryos causes hemorrhages, fatal cardiac defects and embryonic lethality

PubMed Central

Lin, Xiaolin; Zhao, Wentao; Jia, Junshuang; Lin, Taoyan; Xiao, Gaofang; Wang, Shengchun; Lin, Xia; Liu, Yu; Chen, Li; Qin, Yujuan; Li, Jing; Zhang, Tingting; Hao, Weichao; Chen, Bangzhu; Xie, Raoying; Cheng, Yushuang; Xu, Kang; Yao, Kaitai; Huang, Wenhua; Xiao, Dong; Sun, Yan

2016-01-01

Targeted disruption of Cripto-1 in mice caused embryonic lethality at E7.5, whereas we unexpectedly found that ectopic Cripto-1 expression in mouse embryos also led to embryonic lethality, which prompted us to characterize the causes and mechanisms underlying embryonic death due to ectopic Cripto-1 expression. RCLG/EIIa-Cre embryos displayed complex phenotypes between embryonic day 14.5 (E14.5) and E17.5, including fatal hemorrhages (E14.5-E15.5), embryo resorption (E14.5-E17.5), pale body surface (E14.5-E16.5) and no abnormal appearance (E14.5-E16.5). Macroscopic and histological examination revealed that ectopic expression of Cripto-1 transgene in RCLG/EIIa-Cre embryos resulted in lethal cardiac defects, as evidenced by cardiac malformations, myocardial thinning, failed assembly of striated myofibrils and lack of heartbeat. In addition, Cripto-1 transgene activation beginning after E8.5 also caused the aforementioned lethal cardiac defects in mouse embryos. Furthermore, ectopic Cripto-1 expression in embryonic hearts reduced the expression of cardiac transcription factors, which is at least partially responsible for the aforementioned lethal cardiac defects. Our results suggest that hemorrhages and cardiac abnormalities are two important lethal factors in Cripto-1 transgenic mice. Taken together, these findings are the first to demonstrate that sustained Cripto-1 transgene expression after E11.5 causes fatal hemorrhages and lethal cardiac defects, leading to embryonic death at E14.5-17.5. PMID:27687577

Drosophila Araucan and Caupolican Integrate Intrinsic and Signalling Inputs for the Acquisition by Muscle Progenitors of the Lateral Transverse Fate

PubMed Central

Carrasco-Rando, Marta; Tutor, Antonio S.; Prieto-Sánchez, Silvia; González-Pérez, Esther; Barrios, Natalia; Letizia, Annalisa; Martín, Paloma; Campuzano, Sonsoles; Ruiz-Gómez, Mar

2011-01-01

A central issue of myogenesis is the acquisition of identity by individual muscles. In Drosophila, at the time muscle progenitors are singled out, they already express unique combinations of muscle identity genes. This muscle code results from the integration of positional and temporal signalling inputs. Here we identify, by means of loss-of-function and ectopic expression approaches, the Iroquois Complex homeobox genes araucan and caupolican as novel muscle identity genes that confer lateral transverse muscle identity. The acquisition of this fate requires that Araucan/Caupolican repress other muscle identity genes such as slouch and vestigial. In addition, we show that Caupolican-dependent slouch expression depends on the activation state of the Ras/Mitogen Activated Protein Kinase cascade. This provides a comprehensive insight into the way Iroquois genes integrate in muscle progenitors, signalling inputs that modulate gene expression and protein activity. PMID:21811416

Caffeic acid phenethyl ester down-regulates claudin-2 expression at the transcriptional and post-translational levels and enhances chemosensitivity to doxorubicin in lung adenocarcinoma A549 cells.

PubMed

Sonoki, Hiroyuki; Tanimae, Asami; Furuta, Takumi; Endo, Satoshi; Matsunaga, Toshiyuki; Ichihara, Kenji; Ikari, Akira

2018-06-01

Claudin-2 is highly expressed in human lung adenocarcinoma cells and involved in the promotion of proliferation. Here, we searched for a compound, which can decrease claudin-2 expression using lung adenocarcinoma A549 cells. In the screening using compounds included in royal jelly and propolis, the protein level of claudin-2 was dose-dependently decreased by caffeic acid phenethyl ester (CAPE), whereas the mRNA level and promoter activity were only decreased by 50 μM CAPE. These results suggest that CAPE down-regulates claudin-2 expression mediated by two different mechanisms. CAPE (50 μM) decreased the level of p-NF-κB, whereas it increased that of IκB. The CAPE-induced decrease in promoter activity of claudin-2 was blocked by the mutation in an NF-κB-binding site. The inhibition of NF-κB may be involved in the decrease in mRNA level of claudin-2. The CAPE (10 μM)-induced decrease in claudin-2 expression was inhibited by chloroquine, a lysosomal inhibitor. CAPE increased the expression and activity of protein phosphatase (PP) 1 and 2A. The CAPE-induced decrease in claudin-2 expression was blocked by cantharidin, a potent PPs inhibitor. The cell proliferation was suppressed by CAPE, which was partially rescued by ectopic expression of claudin-2. In addition, the toxicity and accumulation of doxorubicin in 3D spheroid cells were enhanced by CAPE, which was inhibited by ectopic expression of claudin-2. Taken together, CAPE down-regulates claudin-2 expression at the transcriptional and post-translational levels, and enhances sensitivity of cells to doxorubicin in 3D culture conditions. CAPE may be a useful adjunctive compound in the treatment of lung adenocarcinoma. Copyright © 2018 Elsevier Inc. All rights reserved.

Mutation of a chitinase-like gene causes ectopic deposition of lignin, aberrant cell shapes, and overproduction of ethylene.

PubMed

Zhong, Ruiqin; Kays, Stanley J; Schroeder, Betty P; Ye, Zheng-Hua

2002-01-01

Chitinase-like proteins have long been proposed to play roles in normal plant growth and development, but no mutations in chitinase-like genes have been obtained previously to support this hypothesis. In this study, we have shown that the gene responsible for the elp1 mutation in Arabidopsis encodes a chitinase-like protein (AtCTL1). Mutation of this chitinase-like gene caused ectopic deposition of lignin and aberrant shapes of cells with incomplete cell walls in the pith of inflorescence stems. The AtCTL1 gene was expressed in all organs during normal plant growth and development, but it was not induced by wounding, salicylic acid, pectin fragments, or ethylene. Consistent with its ubiquitous expression pattern, mutation of the AtCTL1 gene affected many aspects of plant growth and development, including exaggerated hook curvature, reduced length and increased diameter of hypocotyls in dark-grown seedlings, and reduced root length and increased number of root hairs in light-grown seedlings. The mutant phenotypes could be rescued partially by ethylene inhibitors, and ethylene production in the mutant was significantly greater than in the wild type. Together, these results suggest that AtCTL1, a chitinase-like gene, is essential for normal plant growth and development in Arabidopsis.

A thaumatin-like protein of Ocimum basilicum confers tolerance to fungal pathogen and abiotic stress in transgenic Arabidopsis

PubMed Central

Misra, Rajesh Chandra; Sandeep; Kamthan, Mohan; Kumar, Santosh; Ghosh, Sumit

2016-01-01

Plant often responds to fungal pathogens by expressing a group of proteins known as pathogenesis-related proteins (PRs). The expression of PR is mediated through pathogen-induced signal-transduction pathways that are fine-tuned by phytohormones such as methyl jasmonate (MeJA). Here, we report functional characterization of an Ocimum basilicum PR5 family member (ObTLP1) that was identified from a MeJA-responsive expression sequence tag collection. ObTLP1 encodes a 226 amino acid polypeptide that showed sequence and structural similarities with a sweet-tasting protein thaumatin of Thaumatococcus danielli and also with a stress-responsive protein osmotin of Nicotiana tabacum. The expression of ObTLP1 in O. basilicum was found to be organ-preferential under unstressed condition, and responsive to biotic and abiotic stresses, and multiple phytohormone elicitations. Bacterially-expressed recombinant ObTLP1 inhibited mycelial growth of the phytopathogenic fungi, Scleretonia sclerotiorum and Botrytis cinerea; thereby, suggesting its antifungal activity. Ectopic expression of ObTLP1 in Arabidopsis led to enhanced tolerance to S. sclerotiorum and B. cinerea infections, and also to dehydration and salt stress. Moreover, induced expression of the defense marker genes suggested up-regulation of the defense-response pathways in ObTLP1-expressing Arabidopsis upon fungal challenge. Thus, ObTLP1 might be useful for providing tolerance to the fungal pathogens and abiotic stresses in crops. PMID:27150014

A thaumatin-like protein of Ocimum basilicum confers tolerance to fungal pathogen and abiotic stress in transgenic Arabidopsis.

PubMed

Misra, Rajesh Chandra; Sandeep; Kamthan, Mohan; Kumar, Santosh; Ghosh, Sumit

2016-05-06

Plant often responds to fungal pathogens by expressing a group of proteins known as pathogenesis-related proteins (PRs). The expression of PR is mediated through pathogen-induced signal-transduction pathways that are fine-tuned by phytohormones such as methyl jasmonate (MeJA). Here, we report functional characterization of an Ocimum basilicum PR5 family member (ObTLP1) that was identified from a MeJA-responsive expression sequence tag collection. ObTLP1 encodes a 226 amino acid polypeptide that showed sequence and structural similarities with a sweet-tasting protein thaumatin of Thaumatococcus danielli and also with a stress-responsive protein osmotin of Nicotiana tabacum. The expression of ObTLP1 in O. basilicum was found to be organ-preferential under unstressed condition, and responsive to biotic and abiotic stresses, and multiple phytohormone elicitations. Bacterially-expressed recombinant ObTLP1 inhibited mycelial growth of the phytopathogenic fungi, Scleretonia sclerotiorum and Botrytis cinerea; thereby, suggesting its antifungal activity. Ectopic expression of ObTLP1 in Arabidopsis led to enhanced tolerance to S. sclerotiorum and B. cinerea infections, and also to dehydration and salt stress. Moreover, induced expression of the defense marker genes suggested up-regulation of the defense-response pathways in ObTLP1-expressing Arabidopsis upon fungal challenge. Thus, ObTLP1 might be useful for providing tolerance to the fungal pathogens and abiotic stresses in crops.

Zebrafish pronephros tubulogenesis and epithelial identity maintenance are reliant on the polarity proteins Prkc iota and zeta

PubMed Central

Gerlach, Gary F.; Wingert, Rebecca A.

2014-01-01

The zebrafish pronephros provides an excellent in vivo system to study the mechanisms of vertebrate nephron development. When and how renal progenitors in the zebrafish embryo undergo tubulogenesis to form nephrons is poorly understood, but is known to involve a mesenchymal to epithelial transition (MET) and the acquisition of polarity. Here, we determined the precise timing of these events in pronephros tubulogenesis. As the ternary polarity complex is an essential regulator of epithelial cell polarity across tissues, we performed gene knockdown studies to assess the roles of the related factors atypical protein kinase C iota and zeta (prkcι, prkcζ). We found that prkcι and prkcζ serve partially redundant functions to establish pronephros tubule epithelium polarity. Further, the loss of prkcι or the combined knockdown of prkcι/ζ disrupted proximal tubule morphogenesis and podocyte migration due to cardiac defects that prevented normal fluid flow to the kidney. Surprisingly, tubule cells in prkcι/ζ morphants displayed ectopic expression of the transcription factor pax2a and the podocyte-associated genes wt1a, wt1b, and podxl, suggesting that prkcι/ζ are needed to maintain renal epithelial identity. Knockdown of genes essential for cardiac contractility and vascular flow to the kidney, such as tnnt2a, or elimination of pronephros fluid output through knockdown of the intraflagellar transport gene ift88, was not associated with ectopic pronephros gene expression, thus suggesting a unique role for prkcι/ζ in maintaining tubule epithelial identity separate from the consequence of disruptions to renal fluid flow. Interestingly, knockdown of pax2a, but not wt1a, was sufficient to rescue ectopic tubule gene expression in prkcι/ζ morphants. These data suggest a model in which the redundant activities of prkcι and prkcζ are essential to establish tubule epithelial polarity and also serve to maintain proper epithelial cell type identity in the tubule by inhibiting pax2a expression. These studies provide a valuable foundation for further analysis of MET during nephrogenesis, and have implications for understanding the pathways that affect nephron epithelial cells during kidney disease and regeneration. PMID:25446529

Ectopic application of recombinant BMP-2 and BMP-4 can change patterning of developing chick facial primordia.

PubMed

Barlow, A J; Francis-West, P H

1997-01-01

The facial primordia initially consist of buds of undifferentiated mesenchyme, which give rise to a variety of tissues including cartilage, muscle and nerve. These must be arranged in a precise spatial order for correct function. The signals that control facial outgrowth and patterning are largely unknown. The bone morphogenetic proteins Bmp-2 and Bmp-4 are expressed in discrete regions at the distal tips of the early facial primordia suggesting possible roles for BMP-2 and BMP-4 during chick facial development. We show that expression of Bmp-4 and Bmp-2 is correlated with the expression of Msx-1 and Msx-2 and that ectopic application of BMP-2 and BMP-4 can activate Msx-1 and Msx-2 gene expression in the developing facial primordia. We correlate this activation of gene expression with changes in skeletal development. For example, activation of Msx-1 gene expression across the distal tip of the mandibular primordium is associated with an extension of Fgf-4 expression in the epithelium and bifurcation of Meckel's cartilage. In the maxillary primordium, extension of the normal domain of Msx-1 gene expression is correlated with extended epithelial expression of shh and bifurcation of the palatine bone. We also show that application of BMP-2 can increase cell proliferation of the mandibular primordia. Our data suggest that BMP-2 and BMP-4 are part of a signalling cascade that controls outgrowth and patterning of the facial primordia.

Ku proteins function as corepressors to regulate farnesoid X receptor-mediated gene expression

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ohno, Masae; Kunimoto, Masaaki; Nishizuka, Makoto

2009-12-18

The farnesoid X receptor (FXR; NR1H4) is a member of the nuclear receptor superfamily and regulates the expression of genes involved in enterohepatic circulation and the metabolism of bile acids. Based on functional analyses, nuclear receptors are divided into regions A-F. To explore the cofactors interacting with FXR, we performed a pull-down assay using GST-fused to the N-terminal A/B region and the C region, which are required for the ligand-independent transactivation and DNA-binding, respectively, of FXR, and nuclear extracts from HeLa cells. We identified DNA-dependent protein kinase catalytic subunit (DNA-PKcs), Ku80, and Ku70 as FXR associated factors. These proteins aremore » known to have an important role in DNA repair, recombination, and transcription. DNA-PKcs mainly interacted with the A/B region of FXR, whereas the Ku proteins interacted with the C region and with the D region (hinge region). Chromatin immunoprecipitation assays revealed that the Ku proteins associated with FXR on the bile salt export pump (BSEP) promoter. Furthermore, we demonstrated that ectopic expression of the Ku proteins decreased the promoter activity and expression of BSEP gene mediated by FXR. These results suggest that the Ku proteins function as corepressors for FXR.« less

MicroRNA 421 suppresses DPC4/Smad4 in pancreatic cancer

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hao, Jun; Zhang, Shuyu; Zhou, Yingqi

2011-03-25

Research highlights: {yields} We identify miR-421 as a novel potential regulator of DPC4/Smad4. {yields} The expression levels of miR-421 and DPC4/Smad4 are inversely correlated in human clinical specimens of pancreatic cancer. {yields} Overexpression of miR-421 represses the reporter activities driven by the 3'-UTR of DPC4/Smad4 and DPC4/Smad4 protein level in pancreatic cancer cell. {yields} Ectopic expression of miR-421 promotes the proliferation and colony formation of pancreatic cancer cell. -- Abstract: MicroRNAs (miRNAs) have emerged as important regulators in the development of pancreatic cancer and may be a valuable therapeutic application. DPC4/Smad4 is a critical tumor suppressor involved in the progressionmore » of pancreatic cancer, but few studies have been conducted to determine its relationship with miRNAs. In this study, we identify miR-421 as a potential regulator of DPC4/Smad4. We find that in human clinical specimens of pancreatic cancer miR-421 is aberrantly upregulated while DPC4/Smad4 is strongly repressed, and their levels of expression are inversely correlated. Moreover, ectopic expression of miR-421 significantly decreases DPC4/Smad4 protein level in pancreatic cancer cell lines and simultaneously promotes cell proliferation and colony formation in vitro. Our findings identify miR-421 as a potent regulator of DPC4/Smad4, which may provide a novel therapeutic strategy for treatment of DPC4/Smad4-driven pancreatic cancer.« less

Interleukin-18 enhances IL-18R/Nox1 binding, and mediates TRAF3IP2-dependent smooth muscle cell migration. Inhibition by simvastatin

PubMed Central

Valente, Anthony J.; Yoshida, Tadashi; Izadpanah, Reza; Delafontaine, Patrice; Siebenlist, Ulrich; Chandrasekar, Bysani

2013-01-01

We investigated the role of TRAF3 interacting protein 2 (TRAF3IP2), a redox-sensitive adapter protein and an upstream regulator of IKK and JNK in interleukin (IL)-18 induced smooth muscle cell migration, and the mechanism of its inhibition by simvastatin. The pleiotropic cytokine IL-18 induced human coronary artery SMC migration through the induction of TRAF3IP2. IL-18 induced Nox1-dependent ROS generation, TRAF3IP2 expression, and IKK/NF-κB and JNK/AP-1 activation. IL-18 induced its own expression and that of its receptor subunit IL-18Rα. Using co-IP/IB and GST pull-down assays, we show for the first time that the subunits of the IL-18R heterodimer physically associate with Nox1 under basal conditions, and IL-18 appears to enhance their binding. Importantly, the HMG-coA reductase inhibitor simvastatin attenuated IL-18-induced TRAF3IP2 induction. These inhibitory effects were reversed by mevalonate and geranylgeranylpyrophosphate (GGPP), but not by farnesylpyrophosphate (FPP). Interestingly, simvastatin, GGPP, FPP, or Rac1 inhibition did not modulate ectopically expressed TRAF3IP2. The promigratory effects of IL-18 are mediated through TRAF3IP2 in a redox-sensitive manner, and this may involve IL-18R/Nox1 physical association. Further, Simvastatin inhibits inducible, but not ectopically-expressed TRAF3IP2. Targeting TRAF3IP2 may blunt progression of hyperplastic vascular diseases in vivo. PMID:23541442

GPNMB promotes proliferation of developing eosinophils.

PubMed

Hwang, Sae Mi; Kang, Jin Hyun; Kim, Bo Kyum; Uhm, Tae Gi; Kim, Hye Jeong; Lee, Hyune-Hwan; Binas, Bert; Chung, Il Yup

2017-08-01

Glycoprotein non-metastatic melanoma protein B (GPNMB) is a type I transmembrane protein that is expressed in a wide variety of cell types, including haematopoietic lineages. We previously demonstrated that GPNMB is one of the most highly expressed genes at an early and intermediate stage of eosinophil development. We herein examined GPNMB expression and its possible functional effect using cord blood (CB) CD34+ haematopoietic stem cells differentiating toward eosinophils during a 24-day culture period. Western blot and confocal microscopy analyses showed that GPNMB reached its highest levels at day 12 with most GPNMB-positive cells also expressing major basic protein 1 (MBP1), an eosinophil granule protein. GPNMB declined thereafter, but was still present at an appreciable level at day 24, the time when CB eosinophils most abundantly expressed MBP1 and were thus considered fully differentiated. When the developing CB cells were cultured in the presence of a blocking anti-GPNMB antibody, cell proliferation was significantly reduced. In agreement, ectopic expression of GPNMB in heterologous cells resulted in a significant increase in cell proliferation, while small interfering RNA of GPNMB inhibited the GPNMB-mediated proliferation. Thus, GPNMB is expressed in a temporal manner during eosinophil development and delivers a proliferative signal upon activation. © The Authors 2017. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

ATP7A is a novel target of retinoic acid receptor β2 in neuroblastoma cells

PubMed Central

Bohlken, A; Cheung, B B; Bell, J L; Koach, J; Smith, S; Sekyere, E; Thomas, W; Norris, M; Haber, M; Lovejoy, D B; Richardson, D R; Marshall, G M

2009-01-01

Increased retinoic acid receptor β (RARβ2) gene expression is a hallmark of cancer cell responsiveness to retinoid anticancer effects. Moreover, low basal or induced RARβ2 expression is a common feature of many human cancers, suggesting that RARβ2 may act as a tumour suppressor gene in the absence of supplemented retinoid. We have previously shown that low RARβ2 expression is a feature of advanced neuroblastoma. Here, we demonstrate that the ABC domain of the RARβ2 protein alone was sufficient for the growth inhibitory effects of RARβ2 on neuroblastoma cells. ATP7A, the copper efflux pump, is a retinoid-responsive gene, was upregulated by ectopic overexpression of RARβ2. The ectopic overexpression of the RARβ2 ABC domain was sufficient to induce ATP7A expression, whereas, RARβ2 siRNA blocked the induction of ATP7A expression in retinoid-treated neuroblastoma cells. Forced downregulation of ATP7A reduced copper efflux and increased viability of retinoid-treated neuroblastoma cells. Copper supplementation enhanced cell growth and reduced retinoid-responsiveness, whereas copper chelation reduced the viability and proliferative capacity. Taken together, our data demonstrates ATP7A expression is regulated by retinoic acid receptor β and it has effects on intracellular copper levels, revealing a link between the anticancer action of retinoids and copper metabolism. PMID:19127267

Expression of a repressor form of the Arabidopsis thaliana transcription factor TCP16 induces the formation of ectopic meristems.

PubMed

Uberti-Manassero, Nora G; Coscueta, Ezequiel R; Gonzalez, Daniel H

2016-11-01

Plants that express a fusion of the Arabidopsis thaliana class I TCP transcription factor TCP16 to the EAR repressor domain develop several phenotypic alterations, including rounder leaves, short petioles and pedicels, and delayed elongation of sepals, petals and anthers. In addition, these plants develop lobed cotyledons and ectopic meristems. Ectopic meristems are formed on the adaxial side of cotyledon petioles and arise from a cleft that is formed at this site. Analysis of the expression of reporter genes indicated that meristem genes are reactivated at the site of emergence of ectopic meristems, located near the bifurcation of cotyledon veins. The plants also show increased transcript levels of the boundary-specific CUP-SHAPED COTYLEDON (CUC) genes. The results suggest that TCP16 is able to modulate the induction of meristematic programs and the differentiation state of plant cells. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Chemotherapy resistance and metastasis-promoting effects of thyroid hormone in hepatocarcinoma cells are mediated by suppression of FoxO1 and Bim pathway

PubMed Central

Chi, Hsiang-Cheng; Chen, Shen-Liang; Cheng, Yi-Hung; Lin, Tzu-Kang; Tsai, Chung-Ying; Tsai, Ming-Ming; Lin, Yang-Hsiang; Huang, Ya-Hui; Lin, Kwang-Huei

2016-01-01

Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related death worldwide, and systemic chemotherapy is the major treatment strategy for late-stage HCC patients. Poor prognosis following chemotherapy is the general outcome owing to recurrent resistance. Recent studies have suggested that in addition to cytotoxic effects on tumor cells, chemotherapy can induce an alternative cascade that supports tumor growth and metastasis. In the present investigation, we showed that thyroid hormone (TH), a potent hormone-mediating cellular differentiation and metabolism, acts as an antiapoptosis factor upon challenge of thyroid hormone receptor (TR)-expressing HCC cells with cancer therapy drugs, including cisplatin, doxorubicin and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). TH/TR signaling promoted chemotherapy resistance through negatively regulating the pro-apoptotic protein, Bim, resulting in doxorubicin-induced metastasis of chemotherapy-resistant HCC cells. Ectopic expression of Bim in hepatoma cells challenged with chemotherapeutic drugs abolished TH/TR-triggered apoptosis resistance and metastasis. Furthermore, Bim expression was directly transactivated by Forkhead box protein O1 (FoxO1), which was negatively regulated by TH/TR. TH/TR suppressed FoxO1 activity through both transcriptional downregulation and nuclear exclusion of FoxO1 triggered by Akt-mediated phosphorylation. Ectopic expression of the constitutively active FoxO1 mutant, FoxO1-AAA, but not FoxO1-wt, diminished the suppressive effect of TH/TR on Bim. Our findings collectively suggest that expression of Bim is mediated by FoxO1 and indirectly downregulated by TH/TR, leading to chemotherapy resistance and doxorubicin-promoted metastasis of hepatoma cells. PMID:27490929

Cementogenic genes in human periodontal ligament stem cells are downregulated in response to osteogenic stimulation while upregulated by vitamin C treatment

PubMed Central

Gauthier, Philippe; Yu, Zongdong; Tran, Quynh T.; Bhatti, Fazal-Ur-Rehman; Zhu, Xiaofei

2016-01-01

Regeneration of periodontal tissues, particularly cementum, is key to regaining periodontal attachment and health. Human periodontal ligament stem cells (hPDLSCs) have been shown to be a good cell source to regenerate periodontal tissues. However, their subpopulations and the differentiation induction in relation to cementogenic lineages is unclear. Thus, we aim to examine the expression of cementum-associated genes in PDLSC subpopulations and determine the effect of broadly used osteogenic stimulus or vitamin C (VC) on the expression of cementogenic and osteogenic genes in PDLSCs. Our real-time quantitative polymerase chain reaction (qPCR) analysis showed that cementogenic marker cementum attachment protein (CAP) expressed only slightly higher in STRO-1+/CD146+, STRO-1−/CD146+ and STRO-1−/CD146− subpopulations than in the original cell pool, while cementum protein 1 (CEMP1) expression in these subpopulations was not different from the original pool. Notably, under the stimulation with osteogenic differentiation medium, CAP and CEMP1 were down-regulated while osteogenic markers bone sialoprotein (BSP) and osteocalcin (OCN) were upregulated. Both CAP and CEMP1 were upregulated by VC treatment. Transplantation of VC-treated PDLSCs into immunocompromised mice resulted in forming significantly more ectopic cementum- and bone-like mineral tissues in vivo. Immunohistochemical analysis of the ectopic growth showed that CAP and CEMP1 were mainly expressed in the mineral tissue and in some cells of the fibrous tissues. We conclude that osteogenic stimulation is not inductive but appears to be inhibitory of cementogenic pathways, whereas VC induces cementogenic lineage commitment by PDLSCs and may be a useful stimulus for cementogenesis in periodontal regeneration. PMID:27757536

Cementogenic genes in human periodontal ligament stem cells are downregulated in response to osteogenic stimulation while upregulated by vitamin C treatment.

PubMed

Gauthier, Philippe; Yu, Zongdong; Tran, Quynh T; Bhatti, Fazal-Ur-Rehman; Zhu, Xiaofei; Huang, George T-J

2017-04-01

Regeneration of periodontal tissues, particularly cementum, is key to regaining periodontal attachment and health. Human periodontal ligament stem cells (hPDLSCs) have been shown to be a good cell source to regenerate periodontal tissues. However, their subpopulations and the differentiation induction in relation to cementogenic lineages is unclear. Thus, we aim to examine the expression of cementum-associated genes in PDLSC subpopulations and determine the effect of broadly used osteogenic stimulus or vitamin C (VC) on the expression of cementogenic and osteogenic genes in PDLSCs. Our real-time quantitative polymerase chain reaction (qPCR) analysis showed that cementogenic marker cementum attachment protein (CAP) expressed only slightly higher in STRO-1 + /CD146 + , STRO-1 - /CD146 + and STRO-1 - /CD146 - subpopulations than in the original cell pool, while cementum protein 1 (CEMP1) expression in these subpopulations was not different from the original pool. Notably, under the stimulation with osteogenic differentiation medium, CAP and CEMP1 were downregulated while osteogenic markers bone sialoprotein (BSP) and osteocalcin (OCN) were upregulated. Both CAP and CEMP1 were upregulated by VC treatment. Transplantation of VC-treated PDLSCs into immunocompromised mice resulted in forming significantly more ectopic cementum- and bone-like mineral tissues in vivo. Immunohistochemical analysis of the ectopic growth showed that CAP and CEMP1 were mainly expressed in the mineral tissue and in some cells of the fibrous tissues. We conclude that osteogenic stimulation is not inductive but appears to be inhibitory of cementogenic pathways, whereas VC induces cementogenic lineage commitment by PDLSCs and may be a useful stimulus for cementogenesis in periodontal regeneration.

Foxo3 circular RNA promotes cardiac senescence by modulating multiple factors associated with stress and senescence responses.

PubMed

Du, William W; Yang, Weining; Chen, Yu; Wu, Zhong-Kai; Foster, Francis Stuart; Yang, Zhenguo; Li, Xiangmin; Yang, Burton B

2017-05-07

Circular RNAs are a subclass of non-coding RNAs detected within mammalian cells. This study was designed to test the roles of a circular RNA circ-Foxo3 in senescence using in vitro and in vivo approaches. Using the approaches of molecular and cellular biology, we show that a circular RNA generated from a member of the forkhead family of transcription factors, Foxo3, namely circ-Foxo3, was highly expressed in heart samples of aged patients and mice, which was correlated with markers of cellular senescence. Doxorubicin-induced cardiomyopathy was aggravated by ectopic expression of circ-Foxo3 but was relieved by silencing endogenous circ-Foxo3. We also found that silencing circ-Foxo3 inhibited senescence of mouse embryonic fibroblasts and that ectopic expression of circ-Foxo3 induced senescence. We found that circ-Foxo3 was mainly distributed in the cytoplasm, where it interacted with the anti-senescent protein ID-1 and the transcription factor E2F1, as well as the anti-stress proteins FAK and HIF1α. We conclude that ID-1, E2F1, FAK, and HIF1α interact with circ-Foxo3 and are retained in the cytoplasm and could no longer exert their anti-senescent and anti-stress roles, resulting in increased cellular senescence. Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2016. For permissions please email: journals.permissions@oup.com.

Experimental variation of the level and the ratio of angiogenic and osteogenic signaling affects the spatiotemporal expression of bone-specific markers and organization of bone formation in ectopic sites.

PubMed

Moser, Norman; Goldstein, Jan; Kauffmann, Phillip; Epple, Matthias; Schliephake, Henning

2018-04-01

The aim of the present study was to test the hypothesis that the ratio of angiogenic and osteogenic signaling affects ectopic bone formation when delivered in different amounts. Porous composite PDLLA/CaCO 3 scaffolds were loaded with rhBMP2 and rhVEGF in different dosage combinations and implanted into the gluteal muscles of 120 adult male Wistar rats. Bone formation and expression of alkaline phosphatase and Runx2 were quantified by histomorphometry. Spatial distribution across the scaffolds was assessed by using a grid that discriminated between the periphery and center of the scaffolds. The evaluation showed that the combined delivery of bone morphogenetic protein BMP2 and VEGF in different dosage combinations did not enhance the overall quantity of ectopic bone formation compared to the delivery of BMP2 alone. The addition of VEGF generally upregulated Runx2 after 4 weeks, which may have retarded terminal osteogenic differentiation. However, slow combined delivery of 1.5-2.0 μg BMP2 combined with 50 ng VEGF165 over a period of 5 weeks supported a more even distribution of bone formation across the implanted scaffolds whereas higher amounts of VEGF did not elicit this effect. The findings suggest that structural organization rather than the quantity of ectopic bone formation is affected by the dosage and the ratio of BMP2 and VEGF levels at the observed intervals. The development of carriers for dual growth factor delivery has to take into account the necessity to carefully balance the ratio of growth release.

The inhibitor of kappa B kinase-epsilon regulates MMP-3 expression levels and can promote lung metastasis.

PubMed

Seccareccia, E; Pinard, M; Wang, N; Li, S; Burnier, J; Dankort, D; Brodt, P

2014-08-18

The factors that determine the ability of metastatic tumor cells to expand and grow in specific secondary site(s) are not yet fully understood. Matrix metalloproteinases (MMP) were identified as potential regulators of the site-specificity of metastasis. We found that lung carcinoma cells ectopically expressing high levels of the receptor for the type I insulin like growth factor receptor (M27(R) cells) had a significant reduction in MMP-3 expression levels and this coincided with reduced metastasis to the lung. We used these cells to further investigate signaling pathways regulating MMP-3 expression and the role that MMP-3 plays in lung metastasis. We show that ectopic IκB kinase ɛ (IKKɛ) expression in these cells partly restored MMP-3 expression levels and also sensitized MMP-3 transcription to induction by phorbol 12-myristate 13-acetate (PMA). This increase in MMP-3 production was due to increased activation of several signal transduction mediators, including protein kinase C alpha, ERK2, Akt and the transcription factor p65. Furthermore, reconstitution of MMP-3 expression in M27(R) cells restored their ability to colonize the lung whereas silencing of MMP-3 in M27 cells reduced metastases. Collectively, our results implicate IKKɛ as a central regulator of PMA-induced cell signaling and MMP-3 expression and identify MMP-3 as an enabler of tumor cell expansion in the lung.Oncogenesis (2014) 3, e116; doi:10.1038/oncsis.2014.28; published online 18 August 2014.

Differential roles of HIC-5 isoforms in the regulation of cell death and myotube formation during myogenesis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gao Zhengliang; Deblis, Ryan; Glenn, Honor

2007-11-15

Hic-5 is a LIM-Only member of the paxillin superfamily of focal adhesion proteins. It has been shown to regulate a range of biological processes including: senescence, tumorigenesis, steroid hormone action, integrin signaling, differentiation, and apoptosis. To better understand the roles of Hic-5 during development, we initiated a detailed analysis of Hic-5 expression and function in C{sub 2}C{sub 12} myoblasts, a well-established model for myogenesis. We have found that: (1) myoblasts express at least 6 distinct Hic-5 isoforms; (2) the two predominant isoforms, Hic-5{alpha} and Hic-5{beta}, are differentially expressed during myogenesis; (3) any experimentally induced change in Hic-5 expression results inmore » a substantial increase in apoptosis during differentiation; (4) ectopic expression of Hic-5{alpha} is permissive to differentiation while expression of either Hic-5{beta} or antisense Hic-5 blocks myoblast fusion but not chemodifferentiation; (5) Hic-5 localizes to focal adhesions in C{sub 2}C{sub 12} myoblasts and perturbation of Hic-5 leads to defects in cell spreading; (6) alterations in Hic-5 expression interfere with the normal dynamics of laminin expression; and (7) ectopic laminin, but not fibronectin, can rescue the Hic-5-induced blockade of myoblast survival and differentiation. Our data demonstrate differential roles for individual Hic-5 isoforms during myogenesis and support the hypothesis that Hic-5 mediates these effects via integrin signaling.« less

The Effect of Recombinant Tyrosine Hydroxylase Expression on the Neurogenic Differentiation Potency of Mesenchymal Stem Cells

PubMed Central

Duruksu, Gokhan; Karaoz, Erdal

2018-01-01

Objective Tyrosine hydroxylase (TH) is a rate-limiting enzyme in dopamine synthesis, making the enhancement of its activity a target for ensuring sufficient dopamine levels. Rat bone marrow mesenchymal stem cells (rBM-MSCs) are known to synthesize TH after differentiating into neuronal cells through chemical induction, but the effect of its ectopic expression on these cells has not yet been determined. This study investigated the effects of ectopic recombinant TH expression on the stemness characteristics of rBM-MSCs. Methods After cloning, a cell line with stable TH expression was maintained, and the proliferation, the gene expression profile, and differentiation potential of rBM-MSCs were analyzed. Analysis of the cells showed an increment in the proliferation rate that could be reversed by the neutralization of TH. Results The constitutive expression of TH in rBM-MSCs was successfully implemented, without significantly affecting their osteogenic and adipogenic differentiation potential. TH expression improved the expression of other neuronal markers, such as glial fibrillary acidic protein, β-tubulin, nestin, and c-Fos, confirming the neurogenic differentiation capacity of the stem cells. The expression of brain-derived neurotrophic factor (BDNF) and ciliary neurotrophic factor (CNTF) significantly increased after the chemical induction of neurogenic differentiation. Conclusion In this study, the expression of recombinant TH improved the neuroprotective effect of MSCs by upregulating the expression of BDNF and CNTF. Although the neuronal markers were upregulated, the expression of recombinant TH alone in rBM-MSCs was not sufficient for MSCs to differentiate into neurogenic cell lines. PMID:29656620

Differentiation of EL4 lymphoma cells by tumoral environment is associated with inappropriate expression of the large chondroitin sulfate proteoglycan PG-M and the tumor-associated antigen HTgp-175.

PubMed

Rottiers, P; Verfaillie, T; Contreras, R; Revets, H; Desmedt, M; Dooms, H; Fiers, W; Grooten, J

1998-11-09

Progression to malignancy of transformed cells involves complex genetic alterations and aberrant gene expression patterns. While aberrant gene expression is often caused by alterations in individual genes, the contribution of the tumoral environment to the triggering of this gene expression is less well established. The stable but heterogeneous expression in cultured EL4/13 cells of a novel tumor-associated antigen, designated as HTgp-175, was chosen for the investigation of gene expression during tumor formation. Homogeneously HTgp-175-negative EL4/13 cells, isolated by cell sorting or obtained by subcloning, acquired HTgp-175 expression as a result of tumor formation. The tumorigenicity of HTgp-175-negative vs. HTgp-175-positive EL4 variants was identical, indicating that induction but not selection accounted for the phenotypic switch from HTgp-175-negative to HTgp-175-positive. Although mutagenesis experiments showed that the protein was not essential for tumor establishment, tumor-derived cells showed increased malignancy, linking HTgp-175 expression with genetic changes accompanying tumor progression. This novel gene expression was not an isolated event, since it was accompanied by ectopic expression of the large chondroitin sulfate proteoglycan PG-M and of normal differentiation antigens. We conclude that signals derived from the tumoral microenvironment contribute significantly to the aberrant gene expression pattern of malignant cells, apparently by fortuitous activation of differentiation processes and cause expression of novel differentiation antigens as well as of inappropriate tumor-associated and ectopic antigens.

Ectopic lymphoid structures support ongoing production of class-switched autoantibodies in rheumatoid synovium.

PubMed

Humby, Frances; Bombardieri, Michele; Manzo, Antonio; Kelly, Stephen; Blades, Mark C; Kirkham, Bruce; Spencer, Jo; Pitzalis, Costantino

2009-01-13

Follicular structures resembling germinal centres (GCs) that are characterized by follicular dendritic cell (FDC) networks have long been recognized in chronically inflamed tissues in autoimmune diseases, including the synovium of rheumatoid arthritis (RA). However, it is debated whether these ectopic structures promote autoimmunity and chronic inflammation driving the production of pathogenic autoantibodies. Anti-citrullinated protein/peptide antibodies (ACPA) are highly specific markers of RA, predict a poor prognosis, and have been suggested to be pathogenic. Therefore, the main study objectives were to determine whether ectopic lymphoid structures in RA synovium: (i) express activation-induced cytidine deaminase (AID), the enzyme required for somatic hypermutation and class-switch recombination (CSR) of Ig genes; (ii) support ongoing CSR and ACPA production; and (iii) remain functional in a RA/severe combined immunodeficiency (SCID) chimera model devoid of new immune cell influx into the synovium. Using immunohistochemistry (IHC) and quantitative Taqman real-time PCR (QT-PCR) in synovial tissue from 55 patients with RA, we demonstrated that FDC+ structures invariably expressed AID with a distribution resembling secondary lymphoid organs. Further, AID+/CD21+ follicular structures were surrounded by ACPA+/CD138+ plasma cells, as demonstrated by immune reactivity to citrullinated fibrinogen. Moreover, we identified a novel subset of synovial AID+/CD20+ B cells outside GCs resembling interfollicular large B cells. In order to gain direct functional evidence that AID+ structures support CSR and in situ manufacturing of class-switched ACPA, 34 SCID mice were transplanted with RA synovium and humanely killed at 4 wk for harvesting of transplants and sera. Persistent expression of AID and Igamma-Cmu circular transcripts (identifying ongoing IgM-IgG class-switching) was observed in synovial grafts expressing FDCs/CD21L. Furthermore, synovial mRNA levels of AID were closely associated with circulating human IgG ACPA in mouse sera. Finally, the survival and proliferation of functional B cell niches was associated with persistent overexpression of genes regulating ectopic lymphoneogenesis. Our demonstration that FDC+ follicular units invariably express AID and are surrounded by ACPA-producing plasma cells provides strong evidence that ectopic lymphoid structures in the RA synovium are functional and support autoantibody production. This concept is further confirmed by evidence of sustained AID expression, B cell proliferation, ongoing CSR, and production of human IgG ACPA from GC+ synovial tissue transplanted into SCID mice, independently of new B cell influx from the systemic circulation. These data identify AID as a potential therapeutic target in RA and suggest that survival of functional synovial B cell niches may profoundly influence chronic inflammation, autoimmunity, and response to B cell-depleting therapies.

MicroRNA-125b is a novel negative regulator of p53.

PubMed

Le, Minh T N; Teh, Cathleen; Shyh-Chang, Ng; Xie, Huangming; Zhou, Beiyan; Korzh, Vladimir; Lodish, Harvey F; Lim, Bing

2009-04-01

The p53 transcription factor is a key tumor suppressor and a central regulator of the stress response. To ensure a robust and precise response to cellular signals, p53 gene expression must be tightly regulated from the transcriptional to the post-translational levels. Computational predictions suggest that several microRNAs are involved in the post-transcriptional regulation of p53. Here we demonstrate that miR-125b, a brain-enriched microRNA, is a bona fide negative regulator of p53 in both zebrafish and humans. miR-125b-mediated down-regulation of p53 is strictly dependent on the binding of miR-125b to a microRNA response element in the 3' untranslated region of p53 mRNA. Overexpression of miR-125b represses the endogenous level of p53 protein and suppresses apoptosis in human neuroblastoma cells and human lung fibroblast cells. In contrast, knockdown of miR-125b elevates the level of p53 protein and induces apoptosis in human lung fibroblasts and in the zebrafish brain. This phenotype can be rescued significantly by either an ablation of endogenous p53 function or ectopic expression of miR-125b in zebrafish. Interestingly, miR-125b is down-regulated when zebrafish embryos are treated with gamma-irradiation or camptothecin, corresponding to the rapid increase in p53 protein in response to DNA damage. Ectopic expression of miR-125b suppresses the increase of p53 and stress-induced apoptosis. Together, our study demonstrates that miR-125b is an important negative regulator of p53 and p53-induced apoptosis during development and during the stress response.

MicroRNA-125b is a novel negative regulator of p53

PubMed Central

Le, Minh T.N.; Teh, Cathleen; Shyh-Chang, Ng; Xie, Huangming; Zhou, Beiyan; Korzh, Vladimir; Lodish, Harvey F.; Lim, Bing

2009-01-01

The p53 transcription factor is a key tumor suppressor and a central regulator of the stress response. To ensure a robust and precise response to cellular signals, p53 gene expression must be tightly regulated from the transcriptional to the post-translational levels. Computational predictions suggest that several microRNAs are involved in the post-transcriptional regulation of p53. Here we demonstrate that miR-125b, a brain-enriched microRNA, is a bona fide negative regulator of p53 in both zebrafish and humans. miR-125b-mediated down-regulation of p53 is strictly dependent on the binding of miR-125b to a microRNA response element in the 3′ untranslated region of p53 mRNA. Overexpression of miR-125b represses the endogenous level of p53 protein and suppresses apoptosis in human neuroblastoma cells and human lung fibroblast cells. In contrast, knockdown of miR-125b elevates the level of p53 protein and induces apoptosis in human lung fibroblasts and in the zebrafish brain. This phenotype can be rescued significantly by either an ablation of endogenous p53 function or ectopic expression of miR-125b in zebrafish. Interestingly, miR-125b is down-regulated when zebrafish embryos are treated with γ-irradiation or camptothecin, corresponding to the rapid increase in p53 protein in response to DNA damage. Ectopic expression of miR-125b suppresses the increase of p53 and stress-induced apoptosis. Together, our study demonstrates that miR-125b is an important negative regulator of p53 and p53-induced apoptosis during development and during the stress response. PMID:19293287

Short Vegetative Phase-Like MADS-Box Genes Inhibit Floral Meristem Identity in Barley1[W][OA

PubMed Central

Trevaskis, Ben; Tadege, Million; Hemming, Megan N.; Peacock, W. James; Dennis, Elizabeth S.; Sheldon, Candice

2007-01-01

Analysis of the functions of Short Vegetative Phase (SVP)-like MADS-box genes in barley (Hordeum vulgare) indicated a role in determining meristem identity. Three SVP-like genes are expressed in vegetative tissues of barley: Barley MADS1 (BM1), BM10, and Vegetative to Reproductive Transition gene 2. These genes are induced by cold but are repressed during floral development. Ectopic expression of BM1 inhibited spike development and caused floral reversion in barley, with florets at the base of the spike replaced by tillers. Head emergence was delayed in plants that ectopically express BM1, primarily by delayed development after the floral transition, but expression levels of the barley VRN1 gene (HvVRN1) were not affected. Ectopic expression of BM10 inhibited spike development and caused partial floral reversion, where florets at the base of the spike were replaced by inflorescence-like structures, but did not affect heading date. Floral reversion occurred more frequently when BM1 and BM10 ectopic expression lines were grown in short-day conditions. BM1 and BM10 also inhibited floral development and caused floral reversion when expressed in Arabidopsis (Arabidopsis thaliana). We conclude that SVP-like genes function to suppress floral meristem identity in winter cereals. PMID:17114273

Regulatory Circuitry of the CsrA/CsrB and BarA/UvrY Systems of Escherichia coli

PubMed Central

Suzuki, Kazushi; Wang, Xin; Weilbacher, Thomas; Pernestig, Anna-Karin; Melefors, Öjar; Georgellis, Dimitris; Babitzke, Paul; Romeo, Tony

2002-01-01

The global regulator CsrA (carbon storage regulator) is an RNA binding protein that coordinates central carbon metabolism, activates flagellum biosynthesis and motility, and represses biofilm formation in Escherichia coli. CsrA activity is antagonized by the untranslated RNA CsrB, to which it binds and forms a globular ribonucleoprotein complex. CsrA indirectly activates csrB transcription, in an apparent autoregulatory mechanism. In the present study, we elucidate the intermediate regulatory circuitry of this system. Mutations affecting the BarA/UvrY two-component signal transduction system decreased csrB transcription but did not affect csrA′-′lacZ expression. The uvrY defect was severalfold more severe than that of barA. Both csrA and uvrY were required for optimal barA expression. The latter observation suggests an autoregulatory loop for UvrY. Ectopic expression of uvrY suppressed the csrB-lacZ expression defects caused by uvrY, csrA, or barA mutations; csrA suppressed csrA or barA defects; and barA complemented only the barA mutation. Purified UvrY protein stimulated csrB-lacZ expression approximately sixfold in S-30 transcription-translation reactions, revealing a direct effect of UvrY on csrB transcription. Disruption of sdiA, which encodes a LuxR homologue, decreased the expression of uvrY′-′lacZ and csrB-lacZ fusions but did not affect csrA′-′lacZ. The BarA/UvrY system activated biofilm formation. Ectopic expression of uvrY stimulated biofilm formation by a csrB-null mutant, indicative of a CsrB-independent role for UvrY in biofilm development. Collectively, these results demonstrate that uvrY resides downstream from csrA in a signaling pathway for csrB and that CsrA stimulates UvrY-dependent activation of csrB expression by BarA-dependent and -independent mechanisms. PMID:12193630

[Expression of matrix metalloproteinase-1 and tissue inhibitor of metalloproteinase-1 in human and nude mouse ectopic endometrium and the effect of estrogen and progestin on their expression].

PubMed

Lou, Yan-hui; Guo, Xin-hua; Jiang, Hua; Xia, Yu-fang

2010-04-01

To explore the roles of matrix metalloproteinase-1(MMP-1) and tissue inhibitor of metalloproteinase-1(TIMP-1) in the pathogenesis of endometriosis and the effects of estrogen and progestin on their expression. Immunohistochemistry and RT-PCR were employed to detect the expression of MMP-1 and TIMP-1 in the ectopic tissues of 35 patients with endometriosis, 22 eutopic endometrium tissues from women with endometriosis and 28 normal controls. Fifty-nine nude mice were injected with human late secretory endometrial chippings and randomized into estrogen group, progestin group, estrogen-progestin group and control group with corresponding treatments. The implantation rates and graft morphology were observed and MMP-1 and TIMP-1 expressions in the grafts detected by immunohistochemistry. Typical endometrial glands and stroma were observed in all the groups with comparable implantation rates. The administration of progestin was associated with multiple peritoneal implantation sites and significantly larger implants. The transplanted endometria showed proliferative or secretory changes with estrogen or progestin administration. MMP-1 expression significantly increased and TIMP-1 expression decreased with increased MMP-1/TIMP-1 ratio in human and nude mouse ectopic endometria in comparison with those in normal endometria (P<0.05, P<0.01). MMP-1 expression was higher in estrogen and estrogen-progestin groups than in the control group, and was lower in the 3 sexual hormone-treated groups than in the control group. MMP-1 mRNA expression in the eutopic endometrium was significantly higher than that in the normal endometria. Progestrin can not inhibit MMP-1 expression or the effect of estrogen on ectopic endometrium known as progestin resistance. The high expression of MMP-1 and low expression of TIMP-1 in endometriotic tissues confer strong invasiveness of ectopic endometrial tissue, especially in eutopic endometrial tissue, and may play an important role in the pathogenesis of endometriosis.

Cell type-specific roles of Jak3 in IL-2-induced proliferative signal transduction

PubMed Central

Fujii, Hodaka

2007-01-01

Binding of IL-2 to its specific receptor induces activation of two members of Jak family protein tyrosine kinases, Jak1 and Jak3. An IL-2R-reconstituted NIH 3T3 fibroblast cell line proliferates in response to IL-2 only when hematopoietic lineage-specific Jak3 is ectopically expressed. However, the mechanism of Jak3-dependent proliferation in the fibroblast cell line is not known. Here, I showed that Jak3 expression is dispensable for IL-2-induced activation of Jak1 and Stat proteins and expression of nuclear proto-oncogenes in the IL-2R-reconstituted fibroblast cell line. However, Jak3 expression markedly enhanced these IL-2-induced signaling events. In contrast, Jak3 expression was essential for induction of cyclin genes involved in the G1-S transition. These data suggest a critical role of Jak3 in IL-2 signaling in the fibroblast cell line and may provide further insight into the cell type-specific mechanism of cytokine signaling. PMID:17266928

Foxo3 activity promoted by non-coding effects of circular RNA and Foxo3 pseudogene in the inhibition of tumor growth and angiogenesis.

PubMed

Yang, W; Du, W W; Li, X; Yee, A J; Yang, B B

2016-07-28

It has recently been shown that the upregulation of a pseudogene specific to a protein-coding gene could function as a sponge to bind multiple potential targeting microRNAs (miRNAs), resulting in increased gene expression. Similarly, it was recently demonstrated that circular RNAs can function as sponges for miRNAs, and could upregulate expression of mRNAs containing an identical sequence. Furthermore, some mRNAs are now known to not only translate protein, but also function to sponge miRNA binding, facilitating gene expression. Collectively, these appear to be effective mechanisms to ensure gene expression and protein activity. Here we show that expression of a member of the forkhead family of transcription factors, Foxo3, is regulated by the Foxo3 pseudogene (Foxo3P), and Foxo3 circular RNA, both of which bind to eight miRNAs. We found that the ectopic expression of the Foxo3P, Foxo3 circular RNA and Foxo3 mRNA could all suppress tumor growth and cancer cell proliferation and survival. Our results showed that at least three mechanisms are used to ensure protein translation of Foxo3, which reflects an essential role of Foxo3 and its corresponding non-coding RNAs.

Differential protein expression, DNA binding and interaction with SV40 large tumour antigen implicate the p63-family of proteins in replicative senescence.

PubMed

Djelloul, Siham; Tarunina, Marina; Barnouin, Karin; Mackay, Alan; Jat, Parmjit S

2002-02-07

P53 activity plays a key role in mammalian cells when they undergo replicative senescence at their Hayflick limit. To determine whether p63 proteins, members of the family of p53-related genes, are also involved in this process, we examined their expression in serially passaged rat embryo fibroblasts. Upon senescence, two truncated DeltaNp63 proteins decreased in abundance whereas two TAp63 isoforms accumulated. 2-D gel analysis showed that the DeltaNp63 proteins underwent post-translational modifications in both proliferating and senescent cells. Direct binding of DeltaNp63 proteins to a p53 consensus motif was greater in proliferating cells than senescent cells. In contrast p63alpha isoforms bound to DNA in a p53 dependent manner and this was higher in senescent cells than proliferating cells. An interaction of p63alpha proteins with SV40 large tumour antigen was also detected and ectopic expression of DeltaNp63alpha can extend the lifespan of rat embryo fibroblasts. Taken together the results indicate that p63 proteins may play a role in replicative senescence either by competition for p53 DNA binding sites or by direct interaction with p53 protein bound to DNA.

Ectopic Six3 expression in the dragon eye goldfish.

PubMed

Ma, Dong-Mei; Zhu, Hua-Ping; Gui, Jian-Fang

2008-02-01

For goldfish (Carassius auratus), there are many varieties with different eye phenotypes due to artificial selection and adaptive evolution. Dragon eye is a variant eye characterized by a large-size eyeball protruding out of the socket similar to the eye of dragon in Chinese legends. In this study, anatomical structure of the goldfish dragon eye was compared with that of the common eye, and a stretching of the retina was observed in the enlarged dragon eye. Moreover, the homeobox-containing transcription factor Six3 cDNAs were cloned from the two types of goldfish, and the expression patterns were analyzed in both normal eye and dragon eye goldfish. No amino acid sequence differences were observed between the two deduced peptides, and the expression pattern of Six3 protein in dragon eye is quite similar to common eye during embryogenesis, but from 2 days after hatching, ectopic Six3 expression began to occur in the dragon eye, especially in the outer nuclear layer cells. With eye development, more predominant Six3 distribution was detected in the outer nuclear layer cells of dragon eye than that of normal eye, and fewer cell-layers in outer nuclear layer were observed in dragon eye retina than in normal eye retina. The highlight of this study is that higher Six3 expression occurs in dragon eye goldfish than in normal eye goldfish during retinal development of larvae.

Induction of tumor apoptosis through a circular RNA enhancing Foxo3 activity.

PubMed

Du, William W; Fang, Ling; Yang, Weining; Wu, Nan; Awan, Faryal Mehwish; Yang, Zhenguo; Yang, Burton B

2017-02-01

Circular RNAs are a class of non-coding RNAs that are receiving extensive attention. Despite reports showing circular RNAs acting as microRNA sponges, the biological functions of circular RNAs remain largely unknown. We show that in patient tumor samples and in a panel of cancer cells, circ-Foxo3 was minimally expressed. Interestingly, during cancer cell apoptosis, the expression of circ-Foxo3 was found to be significantly increased. We found that silencing endogenous circ-Foxo3 enhanced cell viability, whereas ectopic expression of circ-Foxo3 triggered stress-induced apoptosis and inhibited the growth of tumor xenografts. Also, expression of circ-Foxo3 increased Foxo3 protein levels but repressed p53 levels. By binding to both, circ-Foxo3 promoted MDM2-induced p53 ubiquitination and subsequent degradation, resulting in an overall decrease of p53. With low binding affinity to Foxo3 protein, circ-Foxo3 prevented MDM2 from inducing Foxo3 ubiquitination and degradation, resulting in increased levels of Foxo3 protein. As a result, cell apoptosis was induced by upregulation of the Foxo3 downstream target PUMA.

Gastrokines: stomach-specific proteins with putative homeostatic and tumor suppressor roles.

PubMed

Menheniott, Trevelyan R; Kurklu, Bayzar; Giraud, Andrew S

2013-01-15

During the past decade, a new family of stomach-specific proteins has been recognized. Known as "gastrokines" (GKNs), these secreted proteins are products of gastric mucus-producing cell lineages. GKNs are highly conserved in physical structure, and emerging data point to convergent functions in the modulation of gastric mucosal homeostasis and inflammation. While GKNs are highly prevalent in the normal stomach, frequent loss of GKN expression in gastric cancers, coupled with established antiproliferative activity, suggests putative tumor suppressor roles. Conversely, ectopic expression of GKNs in reparative lesions of Crohn's disease alludes to additional activity in epithelial wound healing and/or repair. Modes of action remain unsolved, but the recent demonstration of a GKN2-trefoil factor 1 heterodimer implicates functional interplay with trefoil factors. This review aims to provide a historical account of GKN biology and encapsulate the rapidly accumulating evidence supporting roles in gastric epithelial homeostasis and tumor suppression.

Ectopic expression of Capsicum-specific cell wall protein Capsicum annuum senescence-delaying 1 (CaSD1) delays senescence and induces trichome formation in Nicotiana benthamiana.

PubMed

Seo, Eunyoung; Yeom, Seon-In; Jo, Sunghwan; Jeong, Heejin; Kang, Byoung-Cheorl; Choi, Doil

2012-04-01

Secreted proteins are known to have multiple roles in plant development, metabolism, and stress response. In a previous study to understand the roles of secreted proteins, Capsicum annuum secreted proteins (CaS) were isolated by yeast secretion trap. Among the secreted proteins, we further characterized Capsicum annuum senescence-delaying 1 (CaSD1), a gene encoding a novel secreted protein that is present only in the genus Capsicum. The deduced CaSD1 contains multiple repeats of the amino acid sequence KPPIHNHKPTDYDRS. Interestingly, the number of repeats varied among cultivars and species in the Capsicum genus. CaSD1 is constitutively expressed in roots, and Agrobacterium-mediated transient overexpression of CaSD1 in Nicotiana benthamiana leaves resulted in delayed senescence with a dramatically increased number of trichomes and enlarged epidermal cells. Furthermore, senescence- and cell division-related genes were differentially regulated by CaSD1-overexpressing plants. These observations imply that the pepper-specific cell wall protein CaSD1 plays roles in plant growth and development by regulating cell division and differentiation.

[Syk inhibitors].

PubMed

Kimura, Yukihiro; Chihara, Kazuyasu; Takeuchi, Kenji; Sada, Kiyonao

2013-07-01

Non-receptor type of protein-tyrosine kinase Syk (spleen tyrosine kinase) was isolated in the University of Fukui in 1991. Syk is known to be essential for the various physiological functions, especially in hematopoietic lineage cells. Moreover, ectopic expression of Syk by epigenetic changes is reported to cause retinoblastoma. Recently, novel Syk inhibitors were developed and its usefulness has been evaluated in the treatment of allergic rhinitis, rheumatoid arthritis, and idiopathic thrombocytopenic purpura. In this review, we will summarize the history, structure, and function of Syk, and then describe the novel Syk inhibitors and their current status. Furthermore, we will introduce our findings of the adaptor protein 3BP2 (c-Abl SH3 domain-binding protein-2), as a novel target of Syk.

Suppression of polyglutamine toxicity by a Drosophila homolog of myeloid leukemia factor 1.

PubMed

Kazemi-Esfarjani, Parsa; Benzer, Seymour

2002-10-01

The toxicity of an abnormally long polyglutamine [poly(Q)] tract within specific proteins is the molecular lesion shared by Huntington's disease (HD) and several other hereditary neurodegenerative disorders. By a genetic screen in Drosophila, devised to uncover genes that suppress poly(Q) toxicity, we discovered a Drosophila homolog of human myeloid leukemia factor 1 (MLF1). Expression of the Drosophila homolog (dMLF) ameliorates the toxicity of poly(Q) expressed in the eye and central nervous system. In the retina, whether endogenously or ectopically expressed, dMLF co-localized with aggregates, suggesting that dMLF alone, or through an intermediary molecular partner, may suppress toxicity by sequestering poly(Q) and/or its aggregates.

Reactive oxygen species contribute to arsenic-induced EZH2 phosphorylation in human bronchial epithelial cells and lung cancer cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Lingzhi; Qiu, Ping; Chen, Bailing

Our previous studies suggested that arsenic is able to induce serine 21 phosphorylation of the EZH2 protein through activation of JNK, STAT3, and Akt signaling pathways in the bronchial epithelial cell line, BEAS-2B. In the present report, we further demonstrated that reactive oxygen species (ROS) were involved in the arsenic-induced protein kinase activation that leads to EZH2 phosphorylation. Several lines of evidence supported this notion. First, the pretreatment of the cells with N-acetyl-L-cysteine (NAC), a potent antioxidant, abolishes arsenic-induced EZH2 phosphorylation along with the inhibition of JNK, STAT3, and Akt. Second, H{sub 2}O{sub 2}, the most important form of ROSmore » in the cells in response to extracellular stress signals, can induce phosphorylation of the EZH2 protein and the activation of JNK, STAT3, and Akt. By ectopic expression of the myc-tagged EZH2, we additionally identified direct interaction and phosphorylation of the EZH2 protein by Akt in response to arsenic and H{sub 2}O{sub 2}. Furthermore, both arsenic and H{sub 2}O{sub 2} were able to induce the translocation of ectopically expressed or endogenous EZH2 from nucleus to cytoplasm. In summary, the data presented in this report indicate that oxidative stress due to ROS generation plays an important role in the arsenic-induced EZH2 phosphorylation. - Highlights:: • Arsenic (As{sup 3+}) induces EZH phosphorylation. • JNK, STAT3, and Akt contribute to EZH2 phosphorylation. • Oxidative stress is involved in As{sup 3+}-induced EZH2 phosphorylation. • As{sup 3+} induces direct interaction of Akt and EZH2. • Phosphorylated EZH2 localized in cytoplasm.« less

Plant mitochondria synthesize melatonin and enhance the tolerance of plants to drought stress.

PubMed

Wang, Lin; Feng, Chao; Zheng, Xiaodong; Guo, Yan; Zhou, Fangfang; Shan, Dongqian; Liu, Xuan; Kong, Jin

2017-10-01

Synthesis of melatonin in mitochondria was reported in animals. However, there is no report on whether plant mitochondria also produce melatonin. Herein, we show that plant mitochondria are a major site for melatonin synthesis. In an in vitro study, isolated apple mitochondria had the capacity to generate melatonin. Subcellular localization analysis documented that an apple SNAT isoform, MzSNAT5, was localized in the mitochondria of both Arabidopsis protoplasts and apple callus cells. The kinetic analysis revealed that the recombinant MzSNAT5 protein exhibited high enzymatic activity to catalyze serotonin to N-acetylserotonin with the K m and V max of 55 μmol/L and 0.909 pmol/min/mg protein at 35°C, respectively; this pathway functioned over a wide range of temperatures from 5 to 75°C. In an in vivo study, MzSNAT5 was drought inducible. The transgenic Arabidopsis ectopically expressing MzSNAT5 elevated the melatonin level and, hence, enhanced drought tolerance. The mechanistic study indicated that the ectopically expressing MzSNAT5 allows plant mitochondria to increase melatonin synthesis. As a potent free radical scavenger, melatonin reduces the oxidative stress caused by the elevated reactive oxygen species which are generated under drought stress in plants. Our findings provide evidence that engineered melatonin-enriched plants exhibit enhanced oxidative tolerance. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Evidence for a functional link between Dd-STATa and Dd-PIAS, a Dictyostelium PIAS homologue.

PubMed

Kawata, Takefumi; Hirano, Tatsunori; Ogasawara, Shun; Aoshima, Ryota; Yachi, Ayako

2011-09-01

Several mammalian protein families inhibit the activity of signal transducer and activator of transcription (STAT) proteins. The protein inhibitor of activated STAT (PIAS) was initially identified through its ability to interact with human STAT proteins. We isolated a gene (pisA) encoding a Dictyostelium orthologue of PIAS, Dd-PIAS, which possesses almost all the representative motifs and domains of mammalian PIAS proteins. A Dd-PIAS null mutant strain displays a normal terminal morphology but with accelerated development once cells are aggregated. In contrast, Dd-PIAS overexpressor strains demonstrate delayed aggregation, almost no slug phototaxis, impaired slug motility, and a prolonged slug migration period. This strain is a near phenocopy of the Dd-STATa null mutant, although it eventually forms a fruiting body, albeit inefficiently. The expression of several Dd-STATa-activated genes is upregulated in the Dd-PIAS null mutant and there is ectopic expression of pstAB makers. The concentration of a PIAS-green fluorescent protein (GFP) fusion protein, expressed under the PIAS promoter, is greatest in the pstO cells and gradually decreases with proximity to the tip of the slug and culminant: a pattern diametrically opposite to that of Dd-STATa. Our results suggest a functional interrelationship between Dd-PIAS and Dd-STATa that influences gene expression and development. © 2011 The Authors. Development, Growth & Differentiation © 2011 Japanese Society of Developmental Biologists.

Amplified RLR signaling activation through an interferon-stimulated gene-endoplasmic reticulum stress-mitochondrial calcium uniporter protein loop

PubMed Central

Cheng, Jinbo; Liao, Yajin; Zhou, Lujun; Peng, Shengyi; Chen, Hong; Yuan, Zengqiang

2016-01-01

Type I interferon (IFN-I) is critical for a host against viral and bacterial infections via induction of hundreds of interferon-stimulated genes (ISGs), but the mechanism underlying the regulation of IFN-I remains largely unknown. In this study, we first demonstrate that ISG expression is required for optimal IFN-β levels, an effect that is further enhanced by endoplasmic reticulum (ER) stress. Furthermore, we identify mitochondrial calcium uniporter protein (MCU) as a mitochondrial antiviral signaling protein (MAVS)-interacting protein that is important for ER stress induction and amplified MAVS signaling activation. In addition, by performing an ectopic expression assay to screen a library of 117 human ISGs for effects on IFN-β levels, we found that tumor necrosis factor receptor 1 (TNFR1) significantly increases IFN-β levels independent of ER stress. Altogether, our findings suggest that MCU and TNFR1 are involved in the regulation of RIG-I-like receptors (RLR) signaling. PMID:26892273

Synaptotagmin 1 causes phosphatidyl inositol lipid-dependent actin remodeling in cultured non-neuronal and neuronal cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Johnsson, Anna-Karin; Karlsson, Roger, E-mail: roger.karlsson@wgi.su.se

2012-01-15

Here we demonstrate that a dramatic actin polymerizing activity caused by ectopic expression of the synaptic vesicle protein synaptotagmin 1 that results in extensive filopodia formation is due to the presence of a lysine rich sequence motif immediately at the cytoplasmic side of the transmembrane domain of the protein. This polybasic sequence interacts with anionic phospholipids in vitro, and, consequently, the actin remodeling caused by this sequence is interfered with by expression of a phosphatidyl inositol (4,5)-bisphosphate (PIP2)-targeted phosphatase, suggesting that it intervenes with the function of PIP2-binding actin control proteins. The activity drastically alters the behavior of a rangemore » of cultured cells including the neuroblastoma cell line SH-SY5Y and primary cortical mouse neurons, and, since the sequence is conserved also in synaptotagmin 2, it may reflect an important fine-tuning role for these two proteins during synaptic vesicle fusion and neurotransmitter release.« less

Sequential and combinatorial roles of maf family genes define proper lens development.

PubMed

Reza, Hasan Mahmud; Urano, Atsuyo; Shimada, Naoko; Yasuda, Kunio

2007-01-16

Maf proteins have been shown to play pivotal roles in lens development in vertebrates. The developing chick lens expresses at least three large Maf proteins. However, the transcriptional relationship among the three large maf genes and their various roles in transactivating the downstream genes largely remain to be elucidated. Chick embryos were electroporated with wild-type L-maf, c-maf, and mafB by in ovo electroporation, and their effects on gene expression were determined by in situ hybridization using specific probes or by immunostaining. Endogenous gene expression was determined using nonelectroporated samples. A regulation mechanism exists among the members of maf family gene. An early-expressed member of this gene family typically stimulates the expression of later-expressed members. We also examined the regulation of various lens-expressing genes with a focus on the interaction between different Maf proteins. We found that the transcriptional ability of Maf proteins varies, even when the target is the same, in parallel with their discrete functions. L-Maf and c-Maf have no effect on E-cadherin expression, whereas MafB enhances its expression and thereby impedes lens vesicle formation. This study also revealed that Maf proteins can regulate the expression of gap junction genes, connexins, and their interacting partner, major intrinsic protein (MIP), during lens development. Misexpression of L-Maf and c-Maf induces ectopic expression of Cx43 and MIP; in contrast, MafB appears to have no effect on Cx43, but induces MIP significantly as evidenced from our gain-of-function experiments. Our results indicate that large Maf function is indispensable for chick lens initiation and development. In addition, L-Maf positively regulates most of the essential genes in this program and directs a series of molecular events leading to proper formation of the lens.

SOX2 plays a critical role in EGFR-mediated self-renewal of human prostate cancer stem-like cells.

PubMed

Rybak, Adrian P; Tang, Damu

2013-12-01

SOX2 is an essential transcription factor for stem cells and plays a role in tumorigenesis, however its role in prostate cancer stem cells (PCSCs) remains unclear. We report here a significant upregulation of SOX2 at both mRNA and protein levels in DU145 PCSCs propagated as suspension spheres in vitro. The expression of SOX2 in DU145 PCSCs is positively regulated by epidermal growth factor receptor (EGFR) signaling. Activation of EGFR signaling, following the addition of epidermal growth factor (EGF) or ectopic expression of a constitutively-active EGFR mutant (EGFRvIII), increased SOX2 expression and the self-renewal of DU145 PCSCs. Conversely, a small molecule EGFR inhibitor (AG1478) blocked EGFR activation, reduced SOX2 expression and inhibited PCSC self-renewal activity, implicating SOX2 in mediating EGFR-dependent self-renewal of PCSCs. In line with this notion, ectopic SOX2 expression enhanced EGF-induced self-renewal of DU145 PCSCs, while SOX2 knockdown reduced PCSC self-renewal with EGF treatment no longer capable of enhancing their propagation. Furthermore, SOX2 knockdown reduced the capacity of DU145 PCSCs to grow under anchorage-independent conditions. Finally, DU145 PCSCs generated xenograft tumors more aggressively with elevated levels of SOX2 expression compared to xenograft tumors derived from non-PCSCs. Collectively, we provide evidence that SOX2 plays a critical role in EGFR-mediated self-renewal of DU145 PCSCs. © 2013.

Hepatitis C virus Core overcomes all-trans retinoic acid-induced apoptosis in human hepatoma cells by inhibiting p14 expression via DNA methylation

PubMed Central

Kwak, Juri; Choi, Jung-Hye; Jang, Kyung Lib

2017-01-01

All-trans retinoic acid (ATRA), the most biologically active metabolite of vitamin A, is known to induce p14 expression via promoter hypomethylation to activate the p14-MDM2-p53 pathway, which leads to activation of the p53-dependent apoptotic pathway and subsequent induction of apoptosis in human hepatoma cells. In the present study, we found that hepatitis C virus (HCV) Core derived from ectopic expression or HCV infection overcomes ATRA-induced apoptosis in p53-positive hepatoma cells. For this effect, HCV Core upregulated both protein levels and enzyme activities of DNA methyltransferase 1 (DNMT1), DNMT3a, and DNMT3b and thereby repressed p14 expression via promoter hypermethylation, resulting in inactivation of the pathway leading to p53 accumulation in the presence of ATRA. As a result, HCV Core prevented ATRA from activating several apoptosis-related molecules, including Bax, p53 upregulated modulator of apoptosis, caspase-9, caspase-3, and poly (ADP-ribose) polymerase. In addition, complementation of p14 in the Core-expressing cells by either ectopic expression or treatment with 5-Aza-2′dC almost completely abolished the potential of HCV Core to suppress ATRA-induced apoptosis. Based on these observations, we conclude that HCV Core executes its oncogenic potential by suppressing the p53-dependent apoptosis induced by ATRA in human hepatoma cells. PMID:29156743

Functional activity and tumor-specific expression of dual oxidase 2 in pancreatic cancer cells and human malignancies characterized with a novel monoclonal antibody.

PubMed

Wu, Yonghzong; Antony, Smitha; Hewitt, Stephen M; Jiang, Guojian; Yang, Sherry X; Meitzler, Jennifer L; Juhasz, Agnes; Lu, Jiamo; Liu, Han; Doroshow, James H; Roy, Krishnendu

2013-04-01

Dual oxidase 2 (Duox2), one of the seven members of the NADPH oxidase gene family, plays a critical role in generating H2O2 for thyroid hormone biosynthesis and as an integral part of the host defense system of the respiratory epithelium and the gastrointestinal tract. Recent evidence suggests that the regulation of Duox2 expression is under the control of pro-inflammatory cytokines and that Duox2-induced reactive oxygen species (ROS) contribute to the inflammation-related tissue injury that occurs in two pre-malignant, inflammatory conditions: chronic pancreatitis and inflammatory bowel disease. Because no reliable Duox antibodies are commercially available, we report the development of a murine monoclonal antibody (MAb) to Duox2 (clone Duox S-12) and its use for the characterization of Duox2 expression in human tumors, tumor cell lines and normal tissues. Duox S-12 specifically detected both endogenously- and ectopically-expressed Duox2 protein by immunoblotting, immunofluorescence microscopy and immunohistochemistry (where both membranous and cytoplasmic staining were present). Duox2 expression detected by Duox S-12 was functionally coupled to the generation of H(2)O(2) in pancreatic cancer cells that expressed Duox2 and its cognate maturation factor DuoxA2. Although Duox S-12 recognizes ectopically expressed Duox1 protein because of the extensive amino acid homology between Duox1 and Duox2, the lack of substantial Duox1 mRNA expression in human tumors (except thyroid cancer) allowed us to evaluate Duox2 expression across a wide range of normal and malignant tissues by immuno-histochemistry. Duox2 was expressed at elevated levels in many human cancers, most notably tumors of the prostate, lung, colon and breast while brain tumors and lymphomas demonstrated the lowest frequency of expression. The Duox-specific monoclonal antibody described here provides a promising tool for the further examination of the role of Duox-dependent reactive oxygen production in inflammation-related carcinogenesis, where alterations in oxidant tone play a critical role in cell growth and proliferation.

Statins Inhibit Monocyte Chemotactic Protein 1 Expression in Endometriosis

PubMed Central

Cakmak, Hakan; Basar, Murat; Seval-Celik, Yasemin; Osteen, Kevin G.; Duleba, Antoni J.; Taylor, Hugh S.; Lockwood, Charles J.; Arici, Aydin

2012-01-01

Statins are potent inhibitors of the endogenous mevalonate pathway. Besides inhibiting cholesterol biosynthesis, statins may also demonstrate anti-inflammatory properties. Inflammation is implicated in the attachment and invasion of endometrial cells to the peritoneal surface and growth of ectopic endometrium by inducing proliferation and angiogenesis. In this study, the effect of statins on monocyte chemotactic protein 1 (MCP-1) expression in endometriotic implants in nude mouse model and in cultured endometriotic cells was evaluated. In mouse model, simvastatin decreased MCP-1 expression in a dose-dependent manner in endometriotic implants (P < .05). Similarly, both simvastatin and mevastatin revealed a dose-dependent inhibition of MCP-1 production in cultured endometriotic cells (P < .01). This inhibitory effect of the statins on MCP-1 production was reversed by the downstream substrates of the mevalonate pathway. Moreover, statins decreased MCP-1 messenger RNA expression in cultured endometriotic cells (P < .05). In conclusion, statins exert anti-inflammatory effect in endometriotic cells and could provide a potential treatment of endometriosis in the future. PMID:22267540

Complete adult neurogenesis within a Wallerian degenerating nerve expressed as an ectopic ganglion.

PubMed

Nakano, Tomonori; Kurimoto, Shigeru; Kato, Shuichi; Asano, Kenichi; Hirata, Takuma; Kiyama, Hiroshi; Hirata, Hitoshi

2018-06-01

Neurogenesis in the adult peripheral nervous system remains to be demonstrated. We transplanted embryonic neural stem cells into a Wallerian degenerating nerve graft and observed development of a nodular structure consisting of neurons, glia, and Schwann cells. Histological analysis revealed a structure loosely resembling the spinal cord, including a synaptic network that formed along the neuron. Furthermore, the new axons reinnervated the paralysed muscle, forming both de novo and revived neuromuscular junctions. Reinnervation of the paralysed muscle resulted in significantly greater mean wet muscle weight and muscle fibre cross-sectional area on the cell transplantation side than on the surgical control side (body weight 0.071 ± 0.011% vs. 0.051 ± 0.007%, p = .006; area 355.6 ± 345.2 vs. 114.0 ± 132.0 μm 2 , p < .001). Electrophysiological experiments demonstrated a functional connection between the neurons and muscle; hence, we identified this nodule as an ectopic ganglion. Surprisingly, in green rat experiments, most of these glial cells, but none of the neurons, expressed enhanced green fluorescent protein, suggesting that the cells constituting the ectopic ganglion were derived from both transplanted stem cells and endogenous stem cells. Such adult neurogenesis in a peripheral nerve related to neural stem cell transplantation has not been reported previously, and these results form the basis for a novel regenerative medicine approach in paralysed muscle. Copyright © 2018 John Wiley & Sons, Ltd.

The cell proliferation antigen Ki-67 organises heterochromatin

PubMed Central

Sobecki, Michal; Mrouj, Karim; Camasses, Alain; Parisis, Nikolaos; Nicolas, Emilien; Llères, David; Gerbe, François; Prieto, Susana; Krasinska, Liliana; David, Alexandre; Eguren, Manuel; Birling, Marie-Christine; Urbach, Serge; Hem, Sonia; Déjardin, Jérôme; Malumbres, Marcos; Jay, Philippe; Dulic, Vjekoslav; Lafontaine, Denis LJ; Feil, Robert; Fisher, Daniel

2016-01-01

Antigen Ki-67 is a nuclear protein expressed in proliferating mammalian cells. It is widely used in cancer histopathology but its functions remain unclear. Here, we show that Ki-67 controls heterochromatin organisation. Altering Ki-67 expression levels did not significantly affect cell proliferation in vivo. Ki-67 mutant mice developed normally and cells lacking Ki-67 proliferated efficiently. Conversely, upregulation of Ki-67 expression in differentiated tissues did not prevent cell cycle arrest. Ki-67 interactors included proteins involved in nucleolar processes and chromatin regulators. Ki-67 depletion disrupted nucleologenesis but did not inhibit pre-rRNA processing. In contrast, it altered gene expression. Ki-67 silencing also had wide-ranging effects on chromatin organisation, disrupting heterochromatin compaction and long-range genomic interactions. Trimethylation of histone H3K9 and H4K20 was relocalised within the nucleus. Finally, overexpression of human or Xenopus Ki-67 induced ectopic heterochromatin formation. Altogether, our results suggest that Ki-67 expression in proliferating cells spatially organises heterochromatin, thereby controlling gene expression. DOI: http://dx.doi.org/10.7554/eLife.13722.001 PMID:26949251

Autoimmune Regulator (AIRE) Is Expressed in Spermatogenic Cells, and It Altered the Expression of Several Nucleic-Acid-Binding and Cytoskeletal Proteins in Germ Cell 1 Spermatogonial (GC1-spg) Cells.

PubMed

Radhakrishnan, Karthika; Bhagya, Kongattu P; Kumar, Anil Tr; Devi, Anandavalli N; Sengottaiyan, Jeeva; Kumar, Pradeep G

2016-08-01

Autoimmune regulator (AIRE) is a gene associated with autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED). AIRE is expressed heavily in the thymic epithelial cells and is involved in maintaining self-tolerance through regulating the expression of tissue-specific antigens. The testes are the most predominant extrathymic location where a heavy expression of AIRE is reported. Homozygous Aire-deficient male mice were infertile, possibly due to impaired spermatogenesis, deregulated germ cell apoptosis, or autoimmunity. We report that AIRE is expressed in the testes of neonatal, adolescent, and adult mice. AIRE expression was detected in glial cell derived neurotrophic factor receptor alpha (GFRα)(+) (spermatogonia), GFRα(-)/synaptonemal complex protein (SCP3)(+) (meiotic), and GFRα(-)/Phosphoglycerate kinase 2 (PGK2)(+) (postmeiotic) germ cells in mouse testes. GC1-spg, a germ-cell-derived cell line, did not express AIRE. Retinoic acid induced AIRE expression in GC1-spg cells. Ectopic expression of AIRE in GC1-spg cells using label-free LC-MS/MS identified a total of 371 proteins that were differentially expressed. 100 proteins were up-regulated, and 271 proteins were down-regulated. Data are available via ProteomeXchange with identifier PXD002511. Functional analysis of the differentially expressed proteins showed increased levels of various nucleic-acid-binding proteins and transcription factors and a decreased level of various cytoskeletal and structural proteins in the AIRE overexpressing cells as compared with the empty vector-transfected controls. The transcripts of a select set of the up-regulated proteins were also elevated. However, there was no corresponding decrease in the mRNA levels of the down-regulated set of proteins. Molecular function network analysis indicated that AIRE influenced gene expression in GC1-spg cells by acting at multiple levels, including transcription, translation, RNA processing, protein transport, protein localization, and protein degradation, thus setting the foundation in understanding the functional role of AIRE in germ cell biology. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Ectopic expression of LEAFY COTYLEDON1-LIKE gene and localized auxin accumulation mark embryogenic competence in epiphyllous plants of Helianthus annuus × H. tuberosus

PubMed Central

Chiappetta, A.; Fambrini, M.; Petrarulo, M.; Rapparini, F.; Michelotti, V.; Bruno, L.; Greco, M.; Baraldi, R.; Salvini, M.; Pugliesi, C.; Bitonti, M. B.

2009-01-01

Background and Aims The clone EMB-2 of the interspecific hybrid Helianthus annuus × H. tuberosus provides an interesting system to study molecular and physiological aspects of somatic embryogenesis. Namely, in addition to non-epiphyllous (NEP) leaves that expand normally, EMB-2 produces epiphyllous (EP) leaves bearing embryos on the adaxial surface. This clone was used to investigate if the ectopic expression of H. annuus LEAFY COTYLEDON1-LIKE (Ha-L1L) gene and auxin activity are correlated with the establishment of embryogenic competence. Methods Ha-L1L expression was evaluated by semi-quantitative RT-PCR and in situ hybridization. The endogenous level and spatial distribution of free indole-3-acetic acid (IAA) were estimated by a capillary gas chromatography–mass spectrometry–selected ion monitoring method and an immuno-cytochemical approach. Key Results Ectopic expression of Ha-L1L was detected in specific cell domains of the adaxial epidermis of EP leaves prior to the development of ectopic embryos. Ha-L1L was expressed rapidly when NEP leaves were induced to regenerate somatic embryos by in vitro culture. Differences in auxin distribution pattern rather than in absolute level were observed between EP and A-2 leaves. More precisely, a strong IAA immuno-signal was detected in single cells or in small groups of cells along the epidermis of EP leaves and accompanied the early stages of embryo development. Changes in auxin level and distribution were observed in NEP leaves induced to regenerate by in vitro culture. Exogenous auxin treatments lightly influenced Ha-L1L transcript levels in spite of an enhancement of the regeneration frequency. Conclusions In EP leaves, Ha-L1L activity marks the putative founder cells of ectopic embryos. Although the ectopic expression of Ha-L1L seems to be not directly mediated by auxin levels per se, it was demonstrated that localized Ha-L1L expression and IAA accumulation in leaf epidermis domains represent early events of somatic embryogenesis displayed by the epiphyllous EMB-2 clone. PMID:19151043

N-MYC down-regulated-like proteins regulate meristem initiation by modulating auxin transport and MAX2 expression.

PubMed

Mudgil, Yashwanti; Ghawana, Sanjay; Jones, Alan M

2013-01-01

N-MYC down-regulated-like (NDL) proteins interact with the Gβ subunit (AGB1) of the heterotrimeric G protein complex and play an important role in AGB1-dependent regulation of lateral root formation by affecting root auxin transport, auxin gradients and the steady-state levels of mRNA encoding the PIN-FORMED 2 and AUXIN 1 auxin transport facilitators. Auxin transport in aerial tissue follows different paths and utilizes different transporters than in roots; therefore, in the present study, we analyzed whether NDL proteins play an important role in AGB1-dependent, auxin-mediated meristem development. Expression levels of NDL gene family members need to be tightly regulated, and altered expression (both over-expression and down-regulation) confers ectopic growth. Over-expression of NDL1 disrupts vegetative and reproductive organ development. Reduced expression of the NDL gene family members results in asymmetric leaf emergence, twinning of rosette leaves, defects in leaf formation, and abnormal silique distribution. Reduced expression of the NDL genes in the agb1-2 (null allele) mutant rescues some of the abnormal phenotypes, such as silique morphology, silique distribution, and peduncle angle, suggesting that proper levels of NDL proteins are maintained by AGB1. We found that all of these abnormal aerial phenotypes due to altered NDL expression were associated with increases in basipetal auxin transport, altered auxin maxima and altered MAX2 expression within the inflorescence stem. NDL proteins, together with AGB1, act as positive regulators of meristem initiation and branching. AGB1 and NDL1 positively regulate basipetal inflorescence auxin transport and modulate MAX2 expression in shoots, which in turn regulates organ and lateral meristem formation by the establishment and maintenance of auxin gradients.

N-MYC DOWN-REGULATED-LIKE Proteins Regulate Meristem Initiation by Modulating Auxin Transport and MAX2 Expression

PubMed Central

Mudgil, Yashwanti; Ghawana, Sanjay; Jones, Alan M.

2013-01-01

Background N-MYC DOWN-REGULATED-LIKE (NDL) proteins interact with the Gβ subunit (AGB1) of the heterotrimeric G protein complex and play an important role in AGB1-dependent regulation of lateral root formation by affecting root auxin transport, auxin gradients and the steady-state levels of mRNA encoding the PIN-FORMED 2 and AUXIN 1 auxin transport facilitators. Auxin transport in aerial tissue follows different paths and utilizes different transporters than in roots; therefore, in the present study, we analyzed whether NDL proteins play an important role in AGB1-dependent, auxin-mediated meristem development. Methodology/Principal Findings Expression levels of NDL gene family members need to be tightly regulated, and altered expression (both over-expression and down-regulation) confers ectopic growth. Over-expression of NDL1 disrupts vegetative and reproductive organ development. Reduced expression of the NDL gene family members results in asymmetric leaf emergence, twinning of rosette leaves, defects in leaf formation, and abnormal silique distribution. Reduced expression of the NDL genes in the agb1-2 (null allele) mutant rescues some of the abnormal phenotypes, such as silique morphology, silique distribution, and peduncle angle, suggesting that proper levels of NDL proteins are maintained by AGB1. We found that all of these abnormal aerial phenotypes due to altered NDL expression were associated with increases in basipetal auxin transport, altered auxin maxima and altered MAX2 expression within the inflorescence stem. Conclusion/Significance NDL proteins, together with AGB1, act as positive regulators of meristem initiation and branching. AGB1 and NDL1 positively regulate basipetal inflorescence auxin transport and modulate MAX2 expression in shoots, which in turn regulates organ and lateral meristem formation by the establishment and maintenance of auxin gradients. PMID:24223735

Activated protein C (APC) can increase bone anabolism via a protease-activated receptor (PAR)1/2 dependent mechanism.

PubMed

Shen, Kaitlin; Murphy, Ciara M; Chan, Ben; Kolind, Mille; Cheng, Tegan L; Mikulec, Kathy; Peacock, Lauren; Xue, Meilang; Park, Sang-Youel; Little, David G; Jackson, Chris J; Schindeler, Aaron

2014-12-01

Activated Protein C (APC) is an anticoagulant with strong cytoprotective properties that has been shown to promote wound healing. In this study APC was investigated for its potential orthopedic application using a Bone Morphogenetic Protein 2 (rhBMP-2) induced ectopic bone formation model. Local co-administration of 10 µg rhBMP-2 with 10 µg or 25 µg APC increased bone volume at 3 weeks by 32% (N.S.) and 74% (p<0.01) compared to rhBMP-2 alone. This was associated with a significant increase in CD31+ and TRAP+ cells in tissue sections of ectopic bone, consistent with enhanced vascularity and bone turnover. The actions of APC are largely mediated by its receptors endothelial protein C receptor (EPCR) and protease-activated receptors (PARs). Cultured pre-osteoblasts and bone nodule tissue sections were shown to express PAR1/2 and EPCR. When pre-osteoblasts were treated with APC, cell viability and phosphorylation of ERK1/2, Akt, and p38 were increased. Inhibition with PAR1 and sometimes PAR2 antagonists, but not with EPCR blocking antibodies, ameliorated the effects of APC on cell viability and kinase phosphorylation. These data indicate that APC can affect osteoblast viability and signaling, and may have in vivo applications with rhBMP-2 for bone repair. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

Abnormal expression of Nrf2 may play an important role in the pathogenesis and development of adenomyosis

PubMed Central

Zhou, Hao; Shen, Fengxian; Li, Juan; Xie, Zhenwei

2017-01-01

Objective To explore the expression level of Nrf2 in adenomyosis and study the mechanism of abnormal expression of Nrf2 in the pathogenesis of adenomyosis. Methods Western blot, immunohistochemistry(IHC) and real time PCR were used to measure Nrf2 expression levels in tissue and cell samples. Knockdown and overexpression of Nrf2 were used to investigate the variation of migration ability of endometrial glandular cells as well as the regulatory mechanism. Results Nrf2 protein levels were significantly higher in the eutopic and ectopic endometrial glands when compared with control cases using IHC and western blot methods. (p< 0.05). However, there was no statistical difference in Nrf2 mRNA expression levels between the adenomyosis and control groups. Using an agonist and Nrf2 siRNA, we regulated the Nrf2 protein levels of primary cultured endometrial glandular cells. With increased expression of Nrf2, cell scratch assay showed that the agonist-treated group migrated significantly faster than the control group, with MMP9 protein level markedly elevated. In contrast, Nrf2 siRNA-treated group migrated slower than the control group, with decreased expression of MMP9 protein. All of the scratching healing spaces and protein levels between the treated and control groups were statistically significant (p< 0.05). Conclusions Abnormal expression of Nrf2 may play an important role in the pathogenesis and development of adenomyosis. Specified reduction of Nrf2 expression could prove to be a new therapeutic target in the clinical treatment of adenomyosis. PMID:28817677

Abnormal expression of Nrf2 may play an important role in the pathogenesis and development of adenomyosis.

PubMed

Chen, Ning; Du, Baoying; Zhou, Hao; Shen, Fengxian; Li, Juan; Xie, Zhenwei

2017-01-01

To explore the expression level of Nrf2 in adenomyosis and study the mechanism of abnormal expression of Nrf2 in the pathogenesis of adenomyosis. Western blot, immunohistochemistry(IHC) and real time PCR were used to measure Nrf2 expression levels in tissue and cell samples. Knockdown and overexpression of Nrf2 were used to investigate the variation of migration ability of endometrial glandular cells as well as the regulatory mechanism. Nrf2 protein levels were significantly higher in the eutopic and ectopic endometrial glands when compared with control cases using IHC and western blot methods. (p< 0.05). However, there was no statistical difference in Nrf2 mRNA expression levels between the adenomyosis and control groups. Using an agonist and Nrf2 siRNA, we regulated the Nrf2 protein levels of primary cultured endometrial glandular cells. With increased expression of Nrf2, cell scratch assay showed that the agonist-treated group migrated significantly faster than the control group, with MMP9 protein level markedly elevated. In contrast, Nrf2 siRNA-treated group migrated slower than the control group, with decreased expression of MMP9 protein. All of the scratching healing spaces and protein levels between the treated and control groups were statistically significant (p< 0.05). Abnormal expression of Nrf2 may play an important role in the pathogenesis and development of adenomyosis. Specified reduction of Nrf2 expression could prove to be a new therapeutic target in the clinical treatment of adenomyosis.

G Protein-Coupled Estrogen Receptor (GPER) Expression in Normal and Abnormal Endometrium

PubMed Central

Lessey, Bruce A.; Taylor, Robert N.; Wang, Wei; Bagchi, Milan K.; Yuan, Lingwen; Scotchie, Jessica; Fritz, Marc A.; Young, Steven L.

2012-01-01

Rapid estrogen effects are mediated by membrane receptors, and evidence suggests a role for both a membrane-associated form of estrogen receptor alpha (ESR1; ERα) and G-protein coupled receptor 30 (GPER; GPR30). Considering estrogen’s importance in endometrial physiology and endometriosis pathophysiology, we hypothesized that GPER could be involved in both cyclic changes in endometrial estrogen action and that aberrant expression might be seen in the eutopic endometrium of women with endometriosis. Using real-time reverse transcriptase–polymerase chain reaction (RT-PCR) and immunohistochemical analysis of normal endometrium, endometrial samples demonstrated cycle-regulated expression of GPER, with maximal expression in the proliferative phase. Eutopic and ectopic endometrium from women with endometriosis overexpressed GPER as compared to eutopic endometrium of normal participants. Ishikawa cells, an adenocarcinoma cell line, expressed GPER, with increased expression upon treatment with estrogen or an ESR1 agonist, but not with a GPER-specific agonist. Decreased expression was seen in Ishikawa cells stably transfected with progesterone receptor A. Together, these data suggest that normal endometrial GPER expression is cyclic and regulated by nuclear estrogen and progesterone receptors, while expression is dysregulated in endometriosis. PMID:22378861

G protein-coupled estrogen receptor (GPER) expression in normal and abnormal endometrium.

PubMed

Plante, Beth J; Lessey, Bruce A; Taylor, Robert N; Wang, Wei; Bagchi, Milan K; Yuan, Lingwen; Scotchie, Jessica; Fritz, Marc A; Young, Steven L

2012-07-01

Rapid estrogen effects are mediated by membrane receptors, and evidence suggests a role for both a membrane-associated form of estrogen receptor alpha (ESR1; ERα) and G-protein coupled receptor 30 (GPER; GPR30). Considering estrogen's importance in endometrial physiology and endometriosis pathophysiology, we hypothesized that GPER could be involved in both cyclic changes in endometrial estrogen action and that aberrant expression might be seen in the eutopic endometrium of women with endometriosis. Using real-time reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemical analysis of normal endometrium, endometrial samples demonstrated cycle-regulated expression of GPER, with maximal expression in the proliferative phase. Eutopic and ectopic endometrium from women with endometriosis overexpressed GPER as compared to eutopic endometrium of normal participants. Ishikawa cells, an adenocarcinoma cell line, expressed GPER, with increased expression upon treatment with estrogen or an ESR1 agonist, but not with a GPER-specific agonist. Decreased expression was seen in Ishikawa cells stably transfected with progesterone receptor A. Together, these data suggest that normal endometrial GPER expression is cyclic and regulated by nuclear estrogen and progesterone receptors, while expression is dysregulated in endometriosis.

MYC-induced cancer cell energy metabolism and therapeutic opportunities.

PubMed

Dang, Chi V; Le, Anne; Gao, Ping

2009-11-01

Although cancers have altered glucose metabolism, termed the Warburg effect, which describes the increased uptake and conversion of glucose to lactate by cancer cells under adequate oxygen tension, changes in the metabolism of glutamine and fatty acid have also been documented. The MYC oncogene, which contributes to the genesis of many human cancers, encodes a transcription factor c-Myc, which links altered cellular metabolism to tumorigenesis. c-Myc regulates genes involved in the biogenesis of ribosomes and mitochondria, and regulation of glucose and glutamine metabolism. With E2F1, c-Myc induces genes involved in nucleotide metabolism and DNA replication, and microRNAs that homeostatically attenuate E2F1 expression. With the hypoxia inducible transcription factor HIF-1, ectopic c-Myc cooperatively induces a transcriptional program for hypoxic adaptation. Myc regulates gene expression either directly, such as glycolytic genes including lactate dehydrogenase A (LDHA), or indirectly, such as repression of microRNAs miR-23a/b to increase glutaminase (GLS) protein expression and glutamine metabolism. Ectopic MYC expression in cancers, therefore, could concurrently drive aerobic glycolysis and/or oxidative phosphorylation to provide sufficient energy and anabolic substrates for cell growth and proliferation in the context of the tumor microenvironment. Collectively, these studies indicate that Myc-mediated altered cancer cell energy metabolism could be translated for the development of new anticancer therapies.

Jaw1/LRMP has a role in maintaining nuclear shape via interaction with SUN proteins.

PubMed

Kozono, Takuma; Tadahira, Kazuko; Okumura, Wataru; Itai, Nao; Tamura-Nakano, Miwa; Dohi, Taeko; Tonozuka, Takashi; Nishikawa, Atsushi

2018-06-06

Jaw1/LRMP is characterized as a type II integral membrane protein that is localized to endoplasmic reticulum (ER), however, its physiological functions have been poorly understood. An alignment of amino acid sequence of Jaw1 with KASH proteins, outer nuclear membrane proteins, revealed that Jaw1 has a partial homology to the KASH domain. Here, we show that the function of Jaw1 is to maintain nuclear shape in mouse melanoma cell line. The siRNA-mediated knockdown of Jaw1 caused a severe defect in nuclear shape, and the defect was rescued by ectopic expression of siRNA-resistant Jaw1. Since co-immunoprecipitation assay indicates that Jaw1 interacts with SUN proteins that are inner nuclear proteins and microtubules, this study suggests that Jaw1 has a role in maintaining nuclear shape via interactions with SUN proteins and microtubules.

Ectopic expression of the HAM59 gene causes homeotic transformations of reproductive organs in sunflower (Helianthus annuus L.).

PubMed

Shulga, O A; Neskorodov, Ya B; Shchennikova, A V; Gaponenko, A K; Skryabin, K G

2015-01-01

The function of the HAM59 MADS-box gene in sunflower (Helianthus annuus L.) was studied to clarify homeotic C activity in the Asteraceae plant family. For the first time, transgenic sunflower plants with a modified pattern of HAM59 expression were obtained. It was shown that the HAM59 MADS-box transcription factor did mediate C activity in sunflower. In particular, it participated in termination of the floral meristem, repression of the cadastral function of A-activity, and together with other C-type sunflower protein HAM45-in the specification of the identity of stamens and pistils.

Zebrafish pronephros tubulogenesis and epithelial identity maintenance are reliant on the polarity proteins Prkc iota and zeta.

PubMed

Gerlach, Gary F; Wingert, Rebecca A

2014-12-15

The zebrafish pronephros provides an excellent in vivo system to study the mechanisms of vertebrate nephron development. When and how renal progenitors in the zebrafish embryo undergo tubulogenesis to form nephrons is poorly understood, but is known to involve a mesenchymal to epithelial transition (MET) and the acquisition of polarity. Here, we determined the precise timing of these events in pronephros tubulogenesis. As the ternary polarity complex is an essential regulator of epithelial cell polarity across tissues, we performed gene knockdown studies to assess the roles of the related factors atypical protein kinase C iota and zeta (prkcι, prkcζ). We found that prkcι and prkcζ serve partially redundant functions to establish pronephros tubule epithelium polarity. Further, the loss of prkcι or the combined knockdown of prkcι/ζ disrupted proximal tubule morphogenesis and podocyte migration due to cardiac defects that prevented normal fluid flow to the kidney. Surprisingly, tubule cells in prkcι/ζ morphants displayed ectopic expression of the transcription factor pax2a and the podocyte-associated genes wt1a, wt1b, and podxl, suggesting that prkcι/ζ are needed to maintain renal epithelial identity. Knockdown of genes essential for cardiac contractility and vascular flow to the kidney, such as tnnt2a, or elimination of pronephros fluid output through knockdown of the intraflagellar transport gene ift88, was not associated with ectopic pronephros gene expression, thus suggesting a unique role for prkcι/ζ in maintaining tubule epithelial identity separate from the consequence of disruptions to renal fluid flow. Interestingly, knockdown of pax2a, but not wt1a, was sufficient to rescue ectopic tubule gene expression in prkcι/ζ morphants. These data suggest a model in which the redundant activities of prkcι and prkcζ are essential to establish tubule epithelial polarity and also serve to maintain proper epithelial cell type identity in the tubule by inhibiting pax2a expression. These studies provide a valuable foundation for further analysis of MET during nephrogenesis, and have implications for understanding the pathways that affect nephron epithelial cells during kidney disease and regeneration. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

Expression of β-catenin protein in hepatocellular carcinoma and its relationship with alpha-fetoprotein.

PubMed

Ren, Ya-Jun; Huang, Tao; Yu, Hong-Lu; Zhang, Li; He, Qian-Jin; Xiong, Zhi-Fan; Peng, Hua

2016-12-01

This study aimed to investigate the expression of β-catenin in hepatocellular carcinoma (HCC) tissues and its relationship with α-fetoprotein (AFP) in HCC. Immunohistochemistry was used to determine the expression of β-catenin in normal liver tissues (n=10), liver cirrhosis tissues (n=20), and primary HCC tissues (n=60). The relationship between β-catenin expression and clinical parameters of HCC was investigated. Real-time PCR and Western blotting were used to detect the mRNA and protein expression levels of β-catenin in the liver cancer cell line SMMC-7721 transfected with a plasmid encoding AFP, and also the mRNA and protein expression levels of β-catenin were measured in the liver cancer cell line Huh7 before and after the transfection with AFP shRNA plasmids. The results showed that β-catenin was only expressed on the cell membrane in normal liver tissues. Its localization to the cytoplasm and nucleus of cells was observed in a small proportion of cirrhotic tissues or adjacent HCC tissues, and such ectopic expression of β-catenin was predominant in HCC tissues. The abnormal expression of β-catenin was correlated with serum AFP levels, cancer cell differentiation and vascular invasion (P<0.05). Additionally, the increased expression of AFP resulted in the upregulation of β-catenin mRNA and protein levels, while knockdown of AFP with AFP shRNA led to significantly decreased β-catenin mRNA and protein levels (P<0.05). It was suggested that the abnormal expression of β-catenin is implicated in hepatic carcinogenesis and development. AFP can lead to increased expression of β-catenin, which may account for the poor prognosis of AFP-associated HCC patients.

Nanobodies and recombinant binders in cell biology

PubMed Central

Helma, Jonas; Cardoso, M. Cristina; Muyldermans, Serge

2015-01-01

Antibodies are key reagents to investigate cellular processes. The development of recombinant antibodies and binders derived from natural protein scaffolds has expanded traditional applications, such as immunofluorescence, binding arrays, and immunoprecipitation. In addition, their small size and high stability in ectopic environments have enabled their use in all areas of cell research, including structural biology, advanced microscopy, and intracellular expression. Understanding these novel reagents as genetic modules that can be integrated into cellular pathways opens up a broad experimental spectrum to monitor and manipulate cellular processes. PMID:26056137

P38 Mitogen-Activated Protein Kinase in Metastasis Associated With Transforming Growth Factor Beta

DTIC Science & Technology

2005-06-01

36, 2001. Shin I, Bakin AV, Rodeck U, Brunet A, Arteaga CL. TGFbeta enhances epithelial cell survival via Akt - dependent regulation of FKHRLI. Mol Biol... Akt mediates cell-cycle progression by phosphorylation of p27Kip’ at threonine 157 and modulation of its cellular localization. Nat Med 8:1145-1152...stress fibers. Ectopic- expression and siRNA experiments show that Smad3 and Smad4 mediate up-regulation of tropomyosins and stress fiber formation

Expression of HOXB2, a retinoic acid signaling target in pancreatic cancer and pancreatic intraepithelial neoplasia.

PubMed

Segara, Davendra; Biankin, Andrew V; Kench, James G; Langusch, Catherine C; Dawson, Amanda C; Skalicky, David A; Gotley, David C; Coleman, Maxwell J; Sutherland, Robert L; Henshall, Susan M

2005-05-01

Despite significant progress in understanding the molecular pathology of pancreatic cancer and its precursor lesion: pancreatic intraepithelial neoplasia (PanIN), there remain no molecules with proven clinical utility as prognostic or therapeutic markers. Here, we used oligonucleotide microarrays to interrogate mRNA expression of pancreatic cancer tissue and normal pancreas to identify novel molecular pathways dysregulated in the development and progression of pancreatic cancer. RNA was hybridized to Affymetrix Genechip HG-U133 oligonucleotide microarrays. A relational database integrating data from publicly available resources was created to identify candidate genes potentially relevant to pancreatic cancer. The protein expression of one candidate, homeobox B2 (HOXB2), in PanIN and pancreatic cancer was assessed using immunohistochemistry. We identified aberrant expression of several components of the retinoic acid (RA) signaling pathway (RARalpha, MUC4, Id-1, MMP9, uPAR, HB-EGF, HOXB6, and HOXB2), many of which are known to be aberrantly expressed in pancreatic cancer and PanIN. HOXB2, a downstream target of RA, was up-regulated 6.7-fold in pancreatic cancer compared with normal pancreas. Immunohistochemistry revealed ectopic expression of HOXB2 in 15% of early PanIN lesions and 48 of 128 (38%) pancreatic cancer specimens. Expression of HOXB2 was associated with nonresectable tumors and was an independent predictor of poor survival in resected tumors. We identified aberrant expression of RA signaling components in pancreatic cancer, including HOXB2, which was expressed in a proportion of PanIN lesions. Ectopic expression of HOXB2 was associated with a poor prognosis for all patients with pancreatic cancer and was an independent predictor of survival in patients who underwent resection.

Acidosis Decreases c-Myc Oncogene Expression in Human Lymphoma Cells: A Role for the Proton-Sensing G Protein-Coupled Receptor TDAG8

PubMed Central

Li, Zhigang; Dong, Lixue; Dean, Eric; Yang, Li V.

2013-01-01

Acidosis is a biochemical hallmark of the tumor microenvironment. Here, we report that acute acidosis decreases c-Myc oncogene expression in U937 human lymphoma cells. The level of c-Myc transcripts, but not mRNA or protein stability, contributes to c-Myc protein reduction under acidosis. The pH-sensing receptor TDAG8 (GPR65) is involved in acidosis-induced c-Myc downregulation. TDAG8 is expressed in U937 lymphoma cells, and the overexpression or knockdown of TDAG8 further decreases or partially rescues c-Myc expression, respectively. Acidic pH alone is insufficient to reduce c-Myc expression, as it does not decrease c-Myc in H1299 lung cancer cells expressing very low levels of pH-sensing G protein-coupled receptors (GPCRs). Instead, c-Myc is slightly increased by acidosis in H1299 cells, but this increase is completely inhibited by ectopic overexpression of TDAG8. Interestingly, TDAG8 expression is decreased by more than 50% in human lymphoma samples in comparison to non-tumorous lymph nodes and spleens, suggesting a potential tumor suppressor function of TDAG8 in lymphoma. Collectively, our results identify a novel mechanism of c-Myc regulation by acidosis in the tumor microenvironment and indicate that modulation of TDAG8 and related pH-sensing receptor pathways may be exploited as a new approach to inhibit Myc expression. PMID:24152439

Polypyrimidine tract-binding protein 1-mediated down-regulation of ATG10 facilitates metastasis of colorectal cancer cells.

PubMed

Jo, Yoon Kyung; Roh, Seon Ae; Lee, Heejin; Park, Na Yeon; Choi, Eun Sun; Oh, Ju-Hee; Park, So Jung; Shin, Ji Hyun; Suh, Young-Ah; Lee, Eun Kyung; Cho, Dong-Hyung; Kim, Jin Cheon

2017-01-28

Autophagy plays complex roles in tumor initiation and development, and the expression of autophagy-related genes (ATGs) is differentially regulated in various cancer cells, depending on their environment. In this study, we analyzed the expressional relationship between polypyrimidine tract-binding protein 1 (PTBP1) and ATG10 in metastatic colorectal cancer. PTBP1 is associated with tumor metastasis in primary colorectal tumors and colorectal cancer liver metastasis (CLM) tissues. In addition, PTPB1 directly interacts with mRNA of ATG10, and regulates ATG10 expression level in colorectal cancer cells. Ectopic expression of PTBP1 decreased ATG10 expression, whereas down-regulation of PTBP1 increased ATG10 level. In contrast to PTBP1, expression of ATG10 was decreased in CLM tissues. Knock down of ATG10 promoted cell migration and invasion of colorectal cancer cells. Moreover, depletion of ATG10 modulated epithelial-mesenchymal transition-associated proteins in colorectal cancer cells: N-cadherin, TCF-8/ZEB1, and CD44 were up-regulated, whereas E-cadherin was down-regulated. Taken together, our findings suggest that expression of ATG10 negatively regulated by PTBP1 is associated with metastasis of colorectal cancer cells. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Hepatic Leukemia Factor Promotes Resistance To Cell Death: Implications For Therapeutics and Chronotherapy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Waters, Katrina M.; Sontag, Ryan L.; Weber, Thomas J.

Physiological variation related to circadian rhythms and aberrant gene expression patterns are believed to modulate therapeutic efficacy, but the precise molecular determinants remain unclear. Here we examine the regulation of cell death by hepatic leukemia factor (HLF), which is an output regulator of circadian rhythms and is aberrantly expressed in human cancers, using an ectopic expression strategy in JB6 mouse epidermal cells and human keratinocytes. Ectopic HLF expression inhibited cell death in both JB6 cells and human keratinocytes, as induced by serum-starvation, tumor necrosis factor alpha and ionizing radiation. Microarray analysis indicates that HLF regulates a complex multi-gene transcriptional programmore » encompassing upregulation of anti-apoptotic genes, downregulation of pro-apoptotic genes, and many additional changes that are consistent with an anti-death program. Collectively, our results demonstrate that ectopic expression of HLF, an established transcription factor that cycles with circadian rhythms, can recapitulate many features associated with circadian-dependent physiological variation.« less

Homologues of the Arabidopsis thaliana SHI/STY/LRP1 genes control auxin biosynthesis and affect growth and development in the moss Physcomitrella patens.

PubMed

Eklund, D Magnus; Thelander, Mattias; Landberg, Katarina; Ståldal, Veronika; Nilsson, Anders; Johansson, Monika; Valsecchi, Isabel; Pederson, Eric R A; Kowalczyk, Mariusz; Ljung, Karin; Ronne, Hans; Sundberg, Eva

2010-04-01

The plant hormone auxin plays fundamental roles in vascular plants. Although exogenous auxin also stimulates developmental transitions and growth in non-vascular plants, the effects of manipulating endogenous auxin levels have thus far not been reported. Here, we have altered the levels and sites of auxin production and accumulation in the moss Physcomitrella patens by changing the expression level of homologues of the Arabidopsis SHI/STY family proteins, which are positive regulators of auxin biosynthesis genes. Constitutive expression of PpSHI1 resulted in elevated auxin levels, increased and ectopic expression of the auxin response reporter GmGH3pro:GUS, and in an increased caulonema/chloronema ratio, an effect also induced by exogenous auxin application. In addition, we observed premature ageing and necrosis in cells ectopically expressing PpSHI1. Knockout of either of the two PpSHI genes resulted in reduced auxin levels and auxin biosynthesis rates in leafy shoots, reduced internode elongation, delayed ageing, a decreased caulonema/chloronema ratio and an increased number of axillary hairs, which constitute potential auxin biosynthesis sites. Some of the identified auxin functions appear to be analogous in vascular and non-vascular plants. Furthermore, the spatiotemporal expression of the PpSHI genes and GmGH3pro:GUS strongly overlap, suggesting that local auxin biosynthesis is important for the regulation of auxin peak formation in non-vascular plants.

Kv10.1 potassium channel: from the brain to the tumors.

PubMed

Cázares-Ordoñez, V; Pardo, L A

2017-10-01

The KCNH1 gene encodes the Kv10.1 (Eag1) ion channel, a member of the EAG (ether-à-go-go) family of voltage-gated potassium channels. Recent studies have demonstrated that KCHN1 mutations are implicated in Temple-Baraitser and Zimmermann-Laband syndromes and other forms of developmental deficits that all present with mental retardation and epilepsy, suggesting that Kv10.1 might be important for cognitive development in humans. Although the Kv10.1 channel is mainly expressed in the mammalian brain, its ectopic expression occurs in 70% of human cancers. Cancer cells and tumors expressing Kv10.1 acquire selective advantages that favor cancer progression through molecular mechanisms that involve several cellular pathways, indicating that protein-protein interactions may be important for Kv10.1 influence in cell proliferation and tumorigenesis. Several studies on transcriptional and post-transcriptional regulation of Kv10.1 expression have shown interesting mechanistic insights about Kv10.1 role in oncogenesis, increasing the importance of identifying the cellular factors that regulate Kv10.1 expression in tumors.

Chicken ovalbumin upstream promoter-transcription factor II regulates nuclear receptor, myogenic, and metabolic gene expression in skeletal muscle cells.

PubMed

Crowther, Lisa M; Wang, Shu-Ching Mary; Eriksson, Natalie A; Myers, Stephen A; Murray, Lauren A; Muscat, George E O

2011-02-24

We demonstrate that chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII) mRNA is more abundantly expressed (than COUP-TFI mRNA) in skeletal muscle C2C12 cells and in (type I and II) skeletal muscle tissue from C57BL/10 mice. Consequently, we have utilized the ABI TaqMan Low Density Array (TLDA) platform to analyze gene expression changes specifically attributable to ectopic COUP-TFII (relative to vector only) expression in muscle cells. Utilizing a TLDA-based platform and 5 internal controls, we analyze the entire NR superfamily, 96 critical metabolic genes, and 48 important myogenic regulatory genes on the TLDA platform utilizing 5 internal controls. The low density arrays were analyzed by rigorous statistical analysis (with Genorm normalization, Bioconductor R, and the Empirical Bayes statistic) using the (integromics) statminer software. In addition, we validated the differentially expressed patho-physiologically relevant gene (identified on the TLDA platform) glucose transporter type 4 (Glut4). We demonstrated that COUP-TFII expression increased the steady state levels of Glut4 mRNA and protein, while ectopic expression of truncated COUP-TFII lacking helix 12 (COUP-TFΔH12) reduced Glut4 mRNA expression in C2C12 cells. Moreover, COUP-TFII expression trans-activated the Glut4 promoter (-997/+3), and ChIP analysis identified selective recruitment of COUP-TFII to a region encompassing a highly conserved SP1 binding site (in mouse, rat, and human) at nt positions -131/-118. Mutation of the SpI site ablated COUP-TFII mediated trans-activation of the Glut4 promoter. In conclusion, this study demonstrates that in skeletal muscle cells, COUP-TFII regulates several nuclear hormone receptors, and critical metabolic and muscle specific genes.

Control of neuronal morphology and connectivity: emerging developmental roles for gap junctional proteins.

PubMed

Baker, Michael W; Macagno, Eduardo R

2014-04-17

Recent evidence indicates that gap junction (GJ) proteins can play a critical role in controlling neuronal connectivity as well as cell morphology in the developing nervous system. GJ proteins may function analogously to cell adhesion molecules, mediating cellular recognition and selective neurite adhesion. Moreover, during synaptogenesis electrical synapses often herald the later establishment of chemical synapses, and thus may help facilitate activity-dependent sculpting of synaptic terminals. Recent findings suggest that the morphology and connectivity of embryonic leech neurons are fundamentally organized by the type and perhaps location of the GJ proteins they express. For example, ectopic expression in embryonic leech neurons of certain innexins that define small GJ-linked networks of cells leads to the novel coupling of the expressing cell into that network. Moreover, gap junctions appear to mediate interactions among homologous neurons that modulate process outgrowth and stability. We propose that the selective formation of GJs between developing neurons and perhaps glial cells in the CNS helps orchestrate not only cellular synaptic connectivity but also can have a pronounced effect on the arborization and morphology of those cells involved. Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Coordinated regulation of neuronal mRNA steady-state levels through developmentally controlled intron retention

PubMed Central

Yap, Karen; Lim, Zhao Qin; Khandelia, Piyush; Friedman, Brad; Makeyev, Eugene V.

2012-01-01

Differentiated cells acquire unique structural and functional traits through coordinated expression of lineage-specific genes. An extensive battery of genes encoding components of the synaptic transmission machinery and specialized cytoskeletal proteins is activated during neurogenesis, but the underlying regulation is not well understood. Here we show that genes encoding critical presynaptic proteins are transcribed at a detectable level in both neurons and nonneuronal cells. However, in nonneuronal cells, the splicing of 3′-terminal introns within these genes is repressed by the polypyrimidine tract-binding protein (Ptbp1). This inhibits the export of incompletely spliced mRNAs to the cytoplasm and triggers their nuclear degradation. Clearance of these intron-containing transcripts occurs independently of the nonsense-mediated decay (NMD) pathway but requires components of the nuclear RNA surveillance machinery, including the nuclear pore-associated protein Tpr and the exosome complex. When Ptbp1 expression decreases during neuronal differentiation, the regulated introns are spliced out, thus allowing the accumulation of translation-competent mRNAs in the cytoplasm. We propose that this mechanism counters ectopic and precocious expression of functionally linked neuron-specific genes and ensures their coherent activation in the appropriate developmental context. PMID:22661231

Coordinated regulation of neuronal mRNA steady-state levels through developmentally controlled intron retention.

PubMed

Yap, Karen; Lim, Zhao Qin; Khandelia, Piyush; Friedman, Brad; Makeyev, Eugene V

2012-06-01

Differentiated cells acquire unique structural and functional traits through coordinated expression of lineage-specific genes. An extensive battery of genes encoding components of the synaptic transmission machinery and specialized cytoskeletal proteins is activated during neurogenesis, but the underlying regulation is not well understood. Here we show that genes encoding critical presynaptic proteins are transcribed at a detectable level in both neurons and nonneuronal cells. However, in nonneuronal cells, the splicing of 3'-terminal introns within these genes is repressed by the polypyrimidine tract-binding protein (Ptbp1). This inhibits the export of incompletely spliced mRNAs to the cytoplasm and triggers their nuclear degradation. Clearance of these intron-containing transcripts occurs independently of the nonsense-mediated decay (NMD) pathway but requires components of the nuclear RNA surveillance machinery, including the nuclear pore-associated protein Tpr and the exosome complex. When Ptbp1 expression decreases during neuronal differentiation, the regulated introns are spliced out, thus allowing the accumulation of translation-competent mRNAs in the cytoplasm. We propose that this mechanism counters ectopic and precocious expression of functionally linked neuron-specific genes and ensures their coherent activation in the appropriate developmental context.

Localization of the RNA-binding proteins Staufen1 and Staufen2 at the mammalian neuromuscular junction.

PubMed

Bélanger, Guy; Stocksley, Mark A; Vandromme, Marie; Schaeffer, Laurent; Furic, Luc; DesGroseillers, Luc; Jasmin, Bernard J

2003-08-01

Staufen is an RNA-binding protein, first identified for its role in oogenesis and CNS development in Drosophila. Two mammalian homologs of Staufen have been identified and shown to bind double-stranded RNA and tubulin, and to function in the somatodendritic transport of mRNA in neurons. Here, we examined whether Staufen proteins are expressed in skeletal muscle in relation to the neuromuscular junction. Immunofluorescence experiments revealed that Staufen1 (Stau1) and Staufen2 (Stau2) accumulate preferentially within the postsynaptic sarcoplasm of muscle fibers as well as at newly formed ectopic synapses. Western blot analyses showed that the levels of Stau1 and Stau2 are greater in slow muscles than in fast-twitch muscles. Muscle denervation induced a significant increase in the expression of Stau1 and Stau2 in the extrasynaptic compartment of both fast and slow muscles. Consistent with these observations, we also demonstrated that expression of Stau1 and Stau2 is increased during myogenic differentiation and that treatment of myotubes with agrin and neuregulin induces a further increase in the expression of both Staufen proteins. We propose that Stau1 and Stau2 are key components of the postsynaptic apparatus in muscle, and that they contribute to the maturation and plasticity of the neuromuscular junction.

The scaffolding and signaling functions of a localization factor impact polar development

PubMed Central

Curtis, Patrick D.; Quardokus, Ellen M.; Lawler, Melanie L.; Guo, Xiaoyun; Klein, David; Chen, Joseph C.; Arnold, Randy J.; Brun, Yves V.

2012-01-01

SUMMARY In the differentiating alphaproteobacterium Caulobacter crescentus, organelle synthesis at cell poles is critical to forming different progeny after cell division. Coordination of polar organelle synthesis, including pili and holdfast, and flagellum ejection, is mediated in part by the scaffolding protein PodJ. At the time of cell division, PodJ undergoes regulated processing to a short form that persists at the flagellar pole of swarmer cells. This study analyzes how PodJ’s role in structural and signaling protein localization impacts organelle synthesis. A PodJ mutant with an internal deletion exhibits reduced sensitivity to pili-tropic phage ΦCbK, resulting from reduced pilA gene expression, which can be linked to altered signaling protein localization. The phage sensitivity defect of a ΔpodJ mutant can be partially suppressed by ectopic pilA expression. Induction of PodJ processing, by manipulation of podJ itself or controlled perP expression, resulted in decreased pilus biogenesis and, when coupled with a podJ mutation that reduced pilA expression, led to complete loss of phage sensitivity. As a whole, the results show that PodJ’s scaffolding role for structural and signaling proteins both contribute to flagellar pole organelle development. PMID:22512778

Human Papillomavirus Types 16 and 18 Early-expressed Proteins Differentially Modulate the Cellular Redox State and DNA Damage

PubMed Central

Cruz-Gregorio, Alfredo; Manzo-Merino, Joaquín; Gonzaléz-García, María Cecilia; Pedraza-Chaverri, José; Medina-Campos, Omar Noel; Valverde, Mahara; Rojas, Emilio; Rodríguez-Sastre, María Alexandra; García-Cuellar, Claudia María; Lizano, Marcela

2018-01-01

Oxidative stress has been proposed as a risk factor for cervical cancer development. However, few studies have evaluated the redox state associated with human papillomavirus (HPV) infection. The aim of this work was to determine the role of the early expressed viral proteins E1, E2, E6 and E7 from HPV types 16 and 18 in the modulation of the redox state in an integral form. Therefore, generation of reactive oxygen species (ROS), concentration of reduced glutathione (GSH), levels and activity of the antioxidant enzymes catalase and superoxide dismutase (SOD) and deoxyribonucleic acid (DNA) damage, were analysed in epithelial cells ectopically expressing the viral proteins. Our research shows that E6 oncoproteins decreased GSH and catalase protein levels, as well as its enzymatic activity, which was associated with an increase in ROS production and DNA damage. In contrast, E7 oncoproteins increased GSH, as well as catalase protein levels and its activity, which correlated with a decrease in ROS without affecting DNA integrity. The co-expression of both E6 and E7 oncoproteins neutralized the effects that were independently observed for each of the viral proteins. Additionally, the combined expression of E1 and E2 proteins increased ROS levels with the subsequent increase in the marker for DNA damage phospho-histone 2AX (γH2AX). A decrease in GSH, as well as SOD2 levels and activity were also detected in the presence of E1 and E2, even though catalase activity increased. This study demonstrates that HPV early expressed proteins differentially modulate cellular redox state and DNA damage. PMID:29483822

Alteration of Nrf2 and Glutamate Cysteine Ligase expression contribute to lesions growth and fibrogenesis in ectopic endometriosis.

PubMed

Marcellin, L; Santulli, P; Chouzenoux, S; Cerles, O; Nicco, C; Dousset, B; Pallardy, M; Kerdine-Römer, S; Just, P A; Chapron, C; Batteux, F

2017-09-01

The redox-sensitive nuclear factor erythroid-derived 2-like 2 (NRF2) controls endogenous antioxidant enzymes' transcription and protects against oxidative damage which is triggered by inflammation and known to favor progression of endometriosis. Glutamate Cysteine Ligase (GCL), a target gene of NRF2, is the first enzyme in the synthesis cascade of glutathione, an important endogenous antioxidant. Sixty-one patients, with thorough surgical examination of the abdominopelvic cavity, were recruited for the study: 31 with histologically-proven endometriosis and 30 disease-free women taken as controls. Expressions of NRF2 and GCL were investigated by quantitative RT-PCR and immunohistochemistry in eutopic and ectopic endometria from endometriosis-affected women and in endometrium of disease-free women. Ex vivo stromal and epithelial cells were extracted and purified from endometrial and endometriotic biopsies to explore expression of NRF2 and GCL in both stromal and epithelial compartments by western blot. Finally, in order to strengthen the role of NRF2 in endometriosis pathogenesis, we evaluated the drop of NRF2 expression in a mouse model of endometriosis using NRF2 knockout (NRF2 -/- ) mice. The mRNA levels of NRF2 and GCL were significantly lower in ectopic endometria of endometriosis-affected women compared to eutopic endometria of disease-free women. The immunohistochemical analysis confirmed the decreased expression of both NRF2 and GCL in ectopic endometriotic tissues compared to eutopic endometria of endometriosis-affected and disease-free women. Immunoblotting revealed a significant decreased of NRF2 and GCL expression in epithelial and stroma cells from ectopic lesions of endometriosis-affected women compared to eutopic endometria from controls. Using a murine model of endometriosis, NRF2 -/- implants were more fibrotic compared to wild-type with an increased weight and volume. These findings indicate that expression of the transcription factor NRF2 and its effector GCL are both profoundly deregulated in endometriotic lesions towards increased growth and fibrogenetic processes. Copyright © 2017 Elsevier Inc. All rights reserved.

Long noncoding RNA H19 interacts with polypyrimidine tract-binding protein 1 to reprogram hepatic lipid homeostasis.

PubMed

Liu, Chune; Yang, Zhihong; Wu, Jianguo; Zhang, Li; Lee, Sangmin; Shin, Dong-Ju; Tran, Melanie; Wang, Li

2018-05-01

H19 is an imprinted long noncoding RNA abundantly expressed in embryonic liver and repressed after birth. We show that H19 serves as a lipid sensor by synergizing with the RNA-binding polypyrimidine tract-binding protein 1 (PTBP1) to modulate hepatic metabolic homeostasis. H19 RNA interacts with PTBP1 to facilitate its association with sterol regulatory element-binding protein 1c mRNA and protein, leading to increased stability and nuclear transcriptional activity. H19 and PTBP1 are up-regulated by fatty acids in hepatocytes and in diet-induced fatty liver, which further augments lipid accumulation. Ectopic expression of H19 induces steatosis and pushes the liver into a "pseudo-fed" state in response to fasting by promoting sterol regulatory element-binding protein 1c protein cleavage and nuclear translocation. Deletion of H19 or knockdown of PTBP1 abolishes high-fat and high-sucrose diet-induced steatosis. Our study unveils an H19/PTBP1/sterol regulatory element-binding protein 1 feedforward amplifying signaling pathway to exacerbate the development of fatty liver. (Hepatology 2018;67:1768-1783). © 2017 by the American Association for the Study of Liver Diseases.

Ectopic Expression of Capsicum-Specific Cell Wall Protein Capsicum annuum Senescence-Delaying 1 (CaSD1) Delays Senescence and Induces Trichome Formation in Nicotiana benthamiana

PubMed Central

Seo, Eunyoung; Yeom, Seon-In; Jo, SungHwan; Jeong, Heejin; Kang, Byoung-Cheorl; Choi, Doil

2012-01-01

Secreted proteins are known to have multiple roles in plant development, metabolism, and stress response. In a previous study to understand the roles of secreted proteins, Capsicum annuum secreted proteins (CaS) were isolated by yeast secretion trap. Among the secreted proteins, we further characterized Capsicum annuum senescence-delaying 1 (CaSD1), a gene encoding a novel secreted protein that is present only in the genus Capsicum. The deduced CaSD1 contains multiple repeats of the amino acid sequence KPPIHNHKPTDYDRS. Interestingly, the number of repeats varied among cultivars and species in the Capsicum genus. CaSD1 is constitutively expressed in roots, and Agrobacterium-mediated transient overexpression of CaSD1 in Nicotiana benthamiana leaves resulted in delayed senescence with a dramatically increased number of trichomes and enlarged epidermal cells. Furthermore, senescence- and cell division-related genes were differentially regulated by CaSD1-overexpressing plants. These observations imply that the pepper-specific cell wall protein CaSD1 plays roles in plant growth and development by regulating cell division and differentiation. PMID:22441673

Compartmentalization of the somite and myogenesis in chick embryos are influenced by wnt expression.

PubMed

Wagner, J; Schmidt, C; Nikowits, W; Christ, B

2000-12-01

Muscles of the body and bones of the axial skeleton derive from specialized regions of somites. Somite development is influenced by adjacent structures. In particular, the dorsal neural tube and the overlying ectoderm have been shown to be necessary for the induction of myogenic precursor cells in the dermomyotome. Members of the Wnt family of signaling molecules, which are expressed in the dorsal neural tube and the ectoderm, are postulated to be responsible for this process. It is shown here that ectopically implanted Wnt-1-, -3a-, and -4-expressing cells alter the process of somite compartmentalization in vivo. An enlarged dorsal compartment results from the implantation of Wnt-expressing cells ventrally between the neural tube/notochord and epithelial somites, at the expense of the ventral compartment, the sclerotome. Thus, ectopic Wnt expression is able to override the influence of ventralizing signals arising from notochord and floor plate. This shift of the border between the two compartments was identified by an increase in the domain of Pax-3 expression and a complete loss of Pax-1 expression in somites close to the ectopic Wnt signal. The expanded expression of MyoD and desmin provides evidence that it is the myotome which increases as a result of Wnt signaling. Paraxis expression is also drastically amplified after implantation of Wnt-expressing cells indicating that Wnts are involved in the formation and maintenance of somite epithelium and suggesting that Paraxis is activated through Wnt signaling pathways. Taken together these results suggest that ectopic Wnts disturb the normal balance of signaling molecules within the somite, resulting in an enhanced recruitment of somitic cells into the myogenic lineage. Copyright 2000 Academic Press.

A Gossypium hirsutum GDSL lipase/hydrolase gene (GhGLIP) appears to be involved in promoting seed growth in Arabidopsis.

PubMed

Ma, Rendi; Yuan, Hali; An, Jing; Hao, Xiaoyun; Li, Hongbin

2018-01-01

GDSL lipase (GLIP) plays a pivotal role in plant cell growth as a multifunctional hydrolytic enzyme. Herein, a cotton (Gossypium hirsutum L. cv Xuzhou 142) GDSL lipase gene (GhGLIP) was obtained from developing ovules and fibers. The GhGLIP cDNA contained an open reading frame (ORF) of 1,143 base pairs (bp) and encodes a putative polypeptide of 380 amino acid residues. Sequence alignment indicated that GhGLIP includes four enzyme catalytic amino acid residue sites of Ser (S), Gly (G), Asn (N) and His (H), located in four conserved blocks. Phylogenetic tree analysis showed that GhGLIP belongs to the typical class IV lipase family with potential functions in plant secondary metabolism. Subcellular distribution analysis demonstrated that GhGLIP localized to the nucleus, cytoplasm and plasma membrane. GhGLIP was expressed predominantly at 5-15 day post anthesis (dpa) in developing ovules and elongating fibers, measured as mRNA levels and enzyme activity. Ectopic overexpression of GhGLIP in Arabidopsis plants resulted in enhanced seed development, including length and fresh weight. Meanwhile, there was increased soluble sugar and protein storage in transgenic Arabidopsis plants, coupled with the promotion of lipase activity. Moreover, the expression of cotton GhGLIP is induced by ethylene (ETH) treatment in vitro. A 1,954-bp GhGLIP promoter was isolated and expressed high activity in driving green fluorescence protein (GFP) expression in tobacco leaves. Cis-acting element analysis of the GhGLIP promoter (pGhGLIP) indicated the presence of an ethylene-responsive element (ERE), and transgenic tobacco leaves with ectopic expression of pGhGLIP::GFP-GUS showed increased GUS activity after ETH treatment. In summary, these results suggest that GhGLIP is a functional enzyme involved in ovule and fiber development and performs significant roles in seed development.

A Gossypium hirsutum GDSL lipase/hydrolase gene (GhGLIP) appears to be involved in promoting seed growth in Arabidopsis

PubMed Central

An, Jing; Hao, Xiaoyun

2018-01-01

GDSL lipase (GLIP) plays a pivotal role in plant cell growth as a multifunctional hydrolytic enzyme. Herein, a cotton (Gossypium hirsutum L. cv Xuzhou 142) GDSL lipase gene (GhGLIP) was obtained from developing ovules and fibers. The GhGLIP cDNA contained an open reading frame (ORF) of 1,143 base pairs (bp) and encodes a putative polypeptide of 380 amino acid residues. Sequence alignment indicated that GhGLIP includes four enzyme catalytic amino acid residue sites of Ser (S), Gly (G), Asn (N) and His (H), located in four conserved blocks. Phylogenetic tree analysis showed that GhGLIP belongs to the typical class IV lipase family with potential functions in plant secondary metabolism. Subcellular distribution analysis demonstrated that GhGLIP localized to the nucleus, cytoplasm and plasma membrane. GhGLIP was expressed predominantly at 5–15 day post anthesis (dpa) in developing ovules and elongating fibers, measured as mRNA levels and enzyme activity. Ectopic overexpression of GhGLIP in Arabidopsis plants resulted in enhanced seed development, including length and fresh weight. Meanwhile, there was increased soluble sugar and protein storage in transgenic Arabidopsis plants, coupled with the promotion of lipase activity. Moreover, the expression of cotton GhGLIP is induced by ethylene (ETH) treatment in vitro. A 1,954-bp GhGLIP promoter was isolated and expressed high activity in driving green fluorescence protein (GFP) expression in tobacco leaves. Cis-acting element analysis of the GhGLIP promoter (pGhGLIP) indicated the presence of an ethylene-responsive element (ERE), and transgenic tobacco leaves with ectopic expression of pGhGLIP::GFP-GUS showed increased GUS activity after ETH treatment. In summary, these results suggest that GhGLIP is a functional enzyme involved in ovule and fiber development and performs significant roles in seed development. PMID:29621331

HIV-1 Tat induces DNMT over-expression through microRNA dysregulation in HIV-related non Hodgkin lymphomas.

PubMed

Luzzi, Anna; Morettini, Federica; Gazaneo, Sara; Mundo, Lucia; Onnis, Anna; Mannucci, Susanna; Rogena, Emily A; Bellan, Cristiana; Leoncini, Lorenzo; De Falco, Giulia

2014-01-01

A close association between HIV infection and the development of cancer exists. Although the advent of highly active antiretroviral therapy has changed the epidemiology of AIDS-associated malignancies, a better understanding on how HIV can induce malignant transformation will help the development of novel therapeutic agents. HIV has been reported to induce the expression of DNMT1 in vitro, but still no information is available about the mechanisms regulating DNMT expression in HIV-related B-cell lymphomas. In this paper, we investigated the expression of DNMT family members (DNMT1, DNMT3a/b) in primary cases of aggressive B-cell lymphomas of HIV-positive subjects. Our results confirmed the activation of DNMT1 by HIV in vivo, and reported for the first time a marked up-regulation of DNMT3a and DNMT3b in HIV-positive aggressive B-cell lymphomas. DNMT up-regulation in HIV-positive tumors correlated with down-regulation of specific microRNAs, as the miR29 family, the miR148-152 cluster, known to regulate their expression. Literature reports the activation of DNMTs by the human polyomavirus BKV large T-antigen and adenovirus E1a, through the pRb/E2F pathway. We have previously demonstrated that the HIV Tat protein is able to bind to the pocket proteins and to inactivate their oncosuppressive properties, resulting in uncontrolled cell proliferation. Therefore, we focused on the role of Tat, due to its capability to be released from infected cells and to dysregulate uninfected ones, using an in vitro model in which Tat was ectopically expressed in B-cells. Our findings demonstrated that the ectopic expression of Tat was per se sufficient to determine DNMT up-regulation, based on microRNA down-regulation, and that this results in aberrant hypermethylation of target genes and microRNAs. These results point at a direct role for Tat in participating in uninfected B-cell lymphomagenesis, through dysregulation of the epigenetical control of gene expression.

Transplantation of autoimmune regulator-encoding bone marrow cells delays the onset of experimental autoimmune encephalomyelitis.

PubMed

Ko, Hyun-Ja; Kinkel, Sarah A; Hubert, François-Xavier; Nasa, Zeyad; Chan, James; Siatskas, Christopher; Hirubalan, Premila; Toh, Ban-Hock; Scott, Hamish S; Alderuccio, Frank

2010-12-01

The autoimmune regulator (AIRE) promotes "promiscuous" expression of tissue-restricted antigens (TRA) in thymic medullary epithelial cells to facilitate thymic deletion of autoreactive T-cells. Here, we show that AIRE-deficient mice showed an earlier development of myelin oligonucleotide glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE). To determine the outcome of ectopic Aire expression, we used a retroviral transduction system to over-express Aire in vitro, in cell lines and in bone marrow (BM). In the cell lines that included those of thymic medullary and dendritic cell origin, ectopically expressed Aire variably promoted expression of TRA including Mog and Ins2 (proII) autoantigens associated, respectively, with the autoimmune diseases multiple sclerosis and type 1 diabetes. BM chimeras generated from BM transduced with a retrovirus encoding Aire displayed elevated levels of Mog and Ins2 expression in thymus and spleen. Following induction of EAE with MOG(35-55), transplanted mice displayed significant delay in the onset of EAE compared with control mice. To our knowledge, this is the first example showing that in vivo ectopic expression of AIRE can modulate TRA expression and alter autoimmune disease development. Copyright © 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Effect of Wnt-1 inducible signaling pathway protein-2 (WISP-2/CCN5), a downstream protein of Wnt signaling, on adipocyte differentiation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Inadera, Hidekuni; Shimomura, Akiko; Tachibana, Shinjiro

2009-02-20

Wnt signaling negatively regulates adipocyte differentiation, and ectopic expression of Wnt-1 in 3T3-L1 cells induces several downstream molecules of Wnt signaling, including Wnt-1 inducible signaling pathway protein (WISP)-2. In this study, we examined the role of WISP-2 in the process of adipocyte differentiation using an in vitro cell culture system. In the differentiation of 3T3-L1 cells, WISP-2 expression was observed in growing cells and declined thereafter. In the mitotic clonal expansion phase of adipocyte differentiation, WISP-2 expression was transiently down-regulated concurrently with up-regulation of CCAAT/enhancer-binding protein {delta} expression. Treatment of 3T3-L1 cells in the differentiation medium with lithium, an activatormore » of Wnt signaling, inhibited the differentiation process with concomitant induction of WISP-2. Treatment of differentiated cells with lithium induced de-differentiation as evidenced by profound reduction of peroxisome proliferator-activator receptor {gamma} expression and concomitant induction of WISP-2. However, de-differentiation of differentiated cells induced by tumor necrosis factor-{alpha} did not induce WISP-2 expression. To directly examine the effect of WISP-2 on adipocyte differentiation, 3T3-L1 cells were infected with a retrovirus carrying WISP-2. Although forced expression of WISP-2 inhibited preadipocyte proliferation, it had no effect on adipocyte differentiation. Thus, although WISP-2 is a downstream protein of Wnt signaling, the role of WISP-2 on adipocyte differentiation may be marginal, at least in this in vitro culture model.« less

MRG-1, an autosome-associated protein, silences X-linked genes and protects germline immortality in Caenorhabditis elegans

PubMed Central

Takasaki, Teruaki; Liu, Zheng; Habara, Yasuaki; Nishiwaki, Kiyoji; Nakayama, Jun-ichi; Inoue, Kunio; Sakamoto, Hiroshi; Strome, Susan

2008-01-01

MRG15, a mammalian protein related to the mortality factor MORF4, is required for cell proliferation and embryo survival. Our genetic analysis has revealed that the Caenorhabditis elegans ortholog MRG-1 serves similar roles. Maternal MRG-1 promotes embryo survival and is required for proliferation and immortality of the primordial germ cells (PGCs). As expected of a chromodomain protein, MRG-1 associates with chromatin. Unexpectedly, it is concentrated on the autosomes and not detectable on the X chromosomes. This association is not dependent on the autosome-enriched protein MES-4. Focusing on possible roles of MRG-1 in regulating gene expression, we determined that MRG-1 is required to maintain repression in the maternal germ line of transgenes on extrachromosomal arrays, and of several X-linked genes previously shown to depend on MES-4 for repression. MRG-1 is not required for PGCs to acquire transcriptional competence or for the turn-on of expression of several PGC-expressed genes (pgl-1, glh-1, glh-4 and nos-1). By contrast to this result in PGCs, MRG-1 is required for ectopic expression of those germline genes in somatic cells lacking the NuRD complex component MEP-1. We discuss how an autosome-enriched protein might repress genes on the X chromosome, promote PGC proliferation and survival, and influence the germ versus soma distinction. PMID:17215300

Functional characterization of an apple apomixis-related MhFIE gene in reproduction development.

PubMed

Liu, Dan-Dan; Dong, Qing-Long; Sun, Chao; Wang, Qing-Lian; You, Chun-Xiang; Yao, Yu-Xin; Hao, Yu-Jin

2012-04-01

The products of the FIS genes play important regulatory roles in diverse developmental processes, especially in seed formation after fertilization. In this study, a FIS-class gene MhFIE was isolated from apple. It encoded a predicted protein highly similar to polycomb group (PcG) protein FERTILIZATION-INDEPENDENT ENDOSPERM (FIE). MhFIE functioned as an Arabidopsis FIE homologue, as indicated by functional complementation experiment using Arabidopsis fie mutant. In addition, BiFC assay showed that MhFIE protein interacted with AtCLF. Furthermore, transgenic Arabidopsis ectopically expressing MhFIE produced less APETALA3 (AtAP3) and AGAMOUS (AtAG) transcripts than WT control, and therefore exhibited abnormal flower, seed development. These results suggested that polycomb complex including FIE and CLF proteins played an important role in reproductive development by regulating the expression of its downstream genes. In addition, it was found that MhFIE constitutively expressed in various tissues tested. Its expression levels were lower in apomictic apple species than the sexual reproductive species, suggested it was possibly involved into apomixis in apple. Furthermore, the hybrids of tea crabapple generated MhFIE transcripts at different levels. The parthenogenesis capacity was negatively correlated with MhFIE expression level in these hybrids. These results suggested that MhFIE was involved into the regulation of flower development and apomixis in apple. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

Alpha-syntrophin deficient mice are protected from adipocyte hypertrophy and ectopic triglyceride deposition in obesity.

PubMed

Eisinger, Kristina; Rein-Fischboeck, Lisa; Neumeier, Markus; Schmidhofer, Sandra; Pohl, Rebekka; Haberl, Elisabeth M; Liebisch, Gerhard; Kopp, Andrea; Schmid, Andreas; Krautbauer, Sabrina; Buechler, Christa

2018-06-01

Alpha-syntrophin (SNTA) is a molecular adapter protein which is expressed in adipocytes. Knock-down of SNTA in 3T3-L1 preadipocytes increases cell proliferation, and differentiated adipocytes display small lipid droplets. These effects are both characteristics of healthy adipose tissue growth which is associated with metabolic improvements in obesity. To evaluate a role of SNTA in adipose tissue morphology and obesity associated metabolic dysfunction, SNTA deficient mice were fed a standard chow or a high fat diet. Mice deficient of SNTA had less fat mass and smaller adipocytes in obesity when compared to control animals. Accordingly, these animals did not develop liver steatosis and did not store excess triglycerides in skeletal muscle upon high fat diet feeding. SNTA-/- animals were protected from hyperinsulinemia and hepatic insulin resistance. Of note, body-weight, food uptake, and serum lipids were normal in the SNTA null mice. SNTA was induced in adipose tissues but not in the liver of diet induced obese and ob/ob mice. In human subcutaneous and visceral fat of seven patients SNTA was similarly expressed and was not associated with body mass index. Current data demonstrate beneficial effects of SNTA deficiency in obesity which is partly attributed to smaller adipocytes and reduced white adipose tissue mass. Higher SNTA protein in fat depots of obese mice may contribute to adipose tissue hypertrophy and ectopic lipid deposition which has to be confirmed in humans. Copyright © 2018 Elsevier Inc. All rights reserved.

A Novel Germline Mutation in BRCA1 Causes Exon 20 Skipping in a Korean Family with a History of Breast Cancer.

PubMed

Yoon, Kyong-Ah; Kong, Sun-Young; Lee, Eun Ji; Cho, Jeong Nam; Chang, Suhwan; Lee, Eun Sook

2017-09-01

Germline mutations in the BRCA1 and BRCA2 genes are strong genetic factors for predispositions to breast, ovarian, and other related cancers. This report describes a family with a history of breast and ovarian cancers that harbored a novel BRCA1 germline mutation. A single nucleotide deletion in intron 20, namely c.5332+4delA, was detected in a 43-year-old patient with breast cancer. This mutation led to the skipping of exon 20, which in turn resulted in the production of a truncated BRCA1 protein that was 1773 amino acids in length. The mother of the proband had died due to ovarian cancer and had harbored the same germline mutation. Ectopically expressed mutant BRCA1 protein interacted with the BARD1 protein, but showed a reduced transcriptional function, as demonstrated by the expression of cyclin B1 . This novel germline mutation in the BRCA1 gene caused familial breast and ovarian cancers.

Tumor Suppressor p53 Stimulates the Expression of Epstein-Barr Virus Latent Membrane Protein 1.

PubMed

Wang, Qianli; Lingel, Amy; Geiser, Vicki; Kwapnoski, Zachary; Zhang, Luwen

2017-10-15

Epstein-Barr virus (EBV) is associated with multiple human malignancies. EBV latent membrane protein 1 (LMP1) is required for the efficient transformation of primary B lymphocytes in vitro and possibly in vivo The tumor suppressor p53 plays a seminal role in cancer development. In some EBV-associated cancers, p53 tends to be wild type and overly expressed; however, the effects of p53 on LMP1 expression is not clear. We find LMP1 expression to be associated with p53 expression in EBV-transformed cells under physiological and DNA damaging conditions. DNA damage stimulates LMP1 expression, and p53 is required for the stimulation. Ectopic p53 stimulates endogenous LMP1 expression. Moreover, endogenous LMP1 blocks DNA damage-mediated apoptosis. Regarding the mechanism of p53-mediated LMP1 expression, we find that interferon regulatory factor 5 (IRF5), a direct target of p53, is associated with both p53 and LMP1. IRF5 binds to and activates a LMP1 promoter reporter construct. Ectopic IRF5 increases the expression of LMP1, while knockdown of IRF5 leads to reduction of LMP1. Furthermore, LMP1 blocks IRF5-mediated apoptosis in EBV-infected cells. All of the data suggest that cellular p53 stimulates viral LMP1 expression, and IRF5 may be one of the factors for p53-mediated LMP1 stimulation. LMP1 may subsequently block DNA damage- and IRF5-mediated apoptosis for the benefits of EBV. The mutual regulation between p53 and LMP1 may play an important role in EBV infection and latency and its related cancers. IMPORTANCE The tumor suppressor p53 is a critical cellular protein in response to various stresses and dictates cells for various responses, including apoptosis. This work suggests that an Epstein-Bar virus (EBV) principal viral oncogene is activated by cellular p53. The viral oncogene blocks p53-mediated adverse effects during viral infection and transformation. Therefore, the induction of the viral oncogene by p53 provides a means for the virus to cope with infection and DNA damage-mediated cellular stresses. This seems to be the first report that p53 activates a viral oncogene; therefore, the discovery would be interesting to a broad readership from the fields of oncology to virology. Copyright © 2017 American Society for Microbiology.

Herpes Simplex Virus 1 Inhibits TANK-Binding Kinase 1 through Formation of the Us11-Hsp90 Complex.

PubMed

Liu, Xing; Main, David; Ma, Yijie; He, Bin

2018-05-09

The Us11 protein of herpes simplex virus 1 (HSV-1) is an accessory factor with multiple functions. In virus-infected cells, it inhibits double-stranded RNA dependent protein kinase PKR, 2',5'-oligoadenylate synthetase, RIG-I and MDA-5. However, its precise role is incompletely defined. By screening human cDNA library, we show that the Us11 protein targets heat shock protein 90 (Hsp90), which inactivates TANK binding kinase 1 (TBK1) and antiviral immunity. When ectopically expressed, HSV-1 Us11 precludes the access of TBK1 to Hsp90 and IFN promoter activation. Consistently, upon HSV infection the Us11 protein suppresses the expression of IFN-β, RANTES, and interferon stimulated genes. This is mirrored by a blockade in the phosphorylation of interferon regulatory factor 3. Mechanistically, the Us11 protein associates with endogenous Hsp90 to disrupt the Hsp90-TBK1 complex. Furthermore, Us11 induces destabilization of TBK1 through a proteasome dependent pathway. Accordingly, Us11 expression facilitates HSV growth. Conversely, TBK1 expression restricts viral replication. These results suggest that control of TBK1 by Us11 promotes HSV-1 infection. IMPORTANCE TANK binding kinase 1 plays a key role in antiviral immunity. Although multiple factors are thought to participate in this process, the picture is obscure in herpes simplex virus infection. We demonstrate that the Us11 protein of HSV-1 forms a complex with heat shock protein 90, which inactivates TANK binding kinase 1 and IFN induction. As a result, expression of the Us11 protein promotes HSV replication. These experimental data provide a new insight into the molecular network of virus-host interactions. Copyright © 2018 American Society for Microbiology.

Variant ionotropic glutamate receptors as chemosensory receptors in Drosophila

PubMed Central

Benton, Richard; Vannice, Kirsten S.; Gomez-Diaz, Carolina; Vosshall, Leslie B.

2009-01-01

Summary Ionotropic glutamate receptors (iGluRs) mediate neuronal communication at synapses throughout vertebrate and invertebrate nervous systems. We have characterized a novel family of iGluR-related genes in Drosophila, which we name Ionotropic Receptors (IRs). These receptors do not belong to the well-described Kainate, AMPA, or NMDA classes of iGluRs, and have divergent ligand-binding domains that lack their characteristic glutamate-interacting residues. IRs are expressed in a combinatorial fashion in sensory neurons that respond to many distinct odors but do not express either insect odorant receptors (ORs) or gustatory receptors (GRs). IR proteins accumulate in sensory dendrites and not at synapses. Mis-expression of IRs induces novel odor responses in ectopic neurons. Together, these results lead us to propose that the IRs comprise a novel family of chemosensory receptors. Conservation of IR/iGluR-related proteins in bacteria, plants, and animals suggests that this receptor family represents an evolutionarily ancient mechanism for sensing both internal and external chemical cues. PMID:19135896

F-box protein FBXL2 targets cyclin D2 for ubiquitination and degradation to inhibit leukemic cell proliferation

PubMed Central

Chen, Bill B.; Glasser, Jennifer R.; Coon, Tiffany A.; Zou, Chunbin; Miller, Hannah L.; Fenton, Moon; McDyer, John F.; Boyiadzis, Michael

2012-01-01

Hematologic maligancies exhibit a growth advantage by up-regulation of components within the molecular apparatus involved in cell-cycle progression. The SCF (Skip-Cullin1-F-box protein) E3 ligase family provides homeostatic feedback control of cell division by mediating ubiquitination and degradation of cell-cycle proteins. By screening several previously undescribed E3 ligase components, we describe the behavior of a relatively new SCF subunit, termed FBXL2, that ubiquitinates and destabilizes cyclin D2 protein leading to G0 phase arrest and apoptosis in leukemic and B-lymphoblastoid cell lines. FBXL2 expression was strongly suppressed, and yet cyclin D2 protein levels were robustly expressed in acute myelogenous leukemia (AML) and acute lymphoblastic leukemia (ALL) patient samples. Depletion of endogenous FBXL2 stabilized cyclin D2 levels, whereas ectopically expressed FBXL2 decreased cyclin D2 lifespan. FBXL2 did not bind a phosphodegron within its substrate, which is typical of other F-box proteins, but uniquely targeted a calmodulin-binding signature within cyclin D2 to facilitate its polyubiquitination. Calmodulin competes with the F-box protein for access to this motif where it bound and protected cyclin D2 from FBXL2. Calmodulin reversed FBXL2-induced G0 phase arrest and attenuated FBXL2-induced apoptosis of lymphoblastoid cells. These results suggest an antiproliferative effect of SCFFBXL2 in lymphoproliferative malignancies. PMID:22323446

Antitumorigenic Effects of ZAKβ, an Alternative Splicing Isoform of ZAK.

PubMed

Lee, Jin-Sun; Lin, Yuh-Yih; Wang, Tsu-Shing; Liu, Jer-Yuh; Lin, Wei-Wen; Yang, Jaw-Ji

2018-02-28

Sterile alpha motif (SAM)- and leucine-zipper-containing kinase (ZAK) plays a role in the regulation of cell cycle progression and oncogenic transformation. The ZAK gene generates two transcript variants, ZAKα and ZAKβ, through alternative splicing. In this study, we identified that ZAKα proteins were upregulated in tumor tissues, whereas ZAKβ proteins were mostly expressed in corresponding normal tissues. The ectopically expressed ZAKβ proteins in cancer cells inhibited cancer cell proliferation as well as anchorage-independent growth. The ZAKβ:ZAKα protein ratio played a role in the regulation of the cyclic adenosine monophosphate (cAMP) signaling pathway, whereas high ZAKβ protein levels led to the activation of cAMP response element binding protein 1 (CREB1) and exerted antitumor properties. Overexpression of ZAKβ or CREB1 cDNAs in cancer cells inhibited anchorage-independent growth and also reduced the levels of cyclooxygenase 2 (Cox2) and β-catenin proteins. Cancer cells treated with doxorubicin (Doxo) resulted in the switching from the expression of ZAKα to ZAKβ and also inhibited cancer cell growth in soft agar, demonstrating that pharmacological drugs could be used to manipulate endogenous reprogramming splicing events and resulting in the activation of endogenous antitumorigenic properties. We showed that the two ZAK transcript variants, ZAKα and ZAKβ, had opposite biological functions in the regulation of tumor cell proliferation in that ZAKβ had powerful antitumor properties and that ZAKα could promote tumor growth.

Effect of hydroxychloroquine and characterization of autophagy in a mouse model of endometriosis

PubMed Central

Ruiz, A; Rockfield, S; Taran, N; Haller, E; Engelman, R W; Flores, I; Panina-Bordignon, P; Nanjundan, M

2016-01-01

In endometriosis, the increased survival potential of shed endometrial cells (which normally undergo anoikis) is suggested to promote lesion development. One mechanism that may alter anoikis is autophagy. Using an autophagic flux inhibitor hydroxychloroquine (HCQ), we identified that it reduces the in vitro survival capacity of human endometriotic and endometrial T-HESC cells. We also identified that HCQ could decrease lesion numbers and disrupt lesion histopathology, as well as increase the levels of peritoneal macrophages and the IP-10 (10 kDa interferon-γ-induced protein) chemokine in a mouse model of endometriosis. We noted that RNA levels of a subset of autophagic markers were reduced in lesions relative to uterine horns from endometriosis-induced (untreated) mice. In addition, the RNA levels of autophagic markers were decreased in uterine horns of endometriosis-induced mice compared with those from controls. However, we noted that protein expression of LC3B (microtubule-associated protein 1 light-chain 3β; an autophagic marker) was increased in uterine horns of endometriosis-induced mice compared with uterine horns of controls. By immunohistochemical staining of a human endometriosis-focused tissue microarray, we observed LC3B expression predominantly in epithelial relative to stromal cells in both eutopic and ectopic endometria. Via transmission electron microscopy, cells from eutopic endometria of endometriosis-induced mice contained more lipid droplets (rather than autophagosomes) compared with uterine horns from controls. Collectively, our findings indicate that the autophagic pathway is dysregulated in both ectopic and eutopic endometrium in a murine model of endometriosis and that HCQ has potential as a therapeutic agent for women afflicted with endometriosis. PMID:26775710

miR-297 modulates multidrug resistance in human colorectal carcinoma by down-regulating MRP-2.

PubMed

Xu, Ke; Liang, Xin; Shen, Ke; Cui, Daling; Zheng, Yuanhong; Xu, Jianhua; Fan, Zhongze; Qiu, Yanyan; Li, Qi; Ni, Lei; Liu, Jianwen

2012-09-01

Colorectal carcinoma is a frequent cause of cancer-related death in men and women. miRNAs (microRNAs) are endogenous small non-coding RNAs that regulate gene expression negatively at the post-transcriptional level. In the present study we investigated the possible role of microRNAs in the development of MDR (multidrug resistance) in colorectal carcinoma cells. We analysed miRNA expression levels between MDR colorectal carcinoma cell line HCT116/L-OHP cells and their parent cell line HCT116 using a miRNA microarray. miR-297 showed lower expression in HCT116/L-OHP cells compared with its parental cells. MRP-2 (MDR-associated protein 2) is an important MDR protein in platinum-drug-resistance cells and is a predicted target of miR-297. Additionally miR-297 was down-regulated in a panel of human colorectal carcinoma tissues and negatively correlated with expression levels of MRP-2. Furthermore, we found that ectopic expression of miR-297 in MDR colorectal carcinoma cells reduced MRP-2 protein level and sensitized these cells to anti-cancer drugs in vitro and in vivo. Taken together, our findings suggest that miR-297 could play a role in the development of MDR in colorectal carcinoma cells, at least in part by modulation of MRP-2.

Ectopic expression of hoxb2 after retinoic acid treatment or mRNA injection: disruption of hindbrain and craniofacial morphogenesis in zebrafish embryos.

PubMed

Yan, Y L; Jowett, T; Postlethwait, J H

1998-12-01

To investigate pattern formation in the vertebrate hindbrain, we isolated a full length hoxb2 cDNA clone from zebrafish. In a gene phylogeny, zebrafish hoxb2 clusters with human HOXB2, and it maps on linkage group 3 along with several other loci whose orthologues are syntenic with human HOXB2. In the hindbrain, hoxb2 is expressed at high levels in rhombomere 3 (r3), lower levels in r4, still lower in r5, and at undetectable levels in r6. In r7, r8, and the rostral spinal cord, hoxb2 is expressed at a lower level than in r5. Lateral cells appearing to emanate from r4 express both hoxb2 and dlx2, suggesting that they are neural crest. Overexpression of hoxb2 by mRNA injections into early cleavage stage embryos resulted in abnormal morphogenesis of the midbrain and rostral hindbrain, abnormal patterning in r4, fusion of cartilage elements arising from pharyngeal arches 1 and 2, and ectopic expression of krx20 and valentino (but not pax2, rtk1, or hoxb1) in the rostral hindbrain, midbrain, and, surprisingly, the eye. Treatments with retinoic acid produced a phenotype similar to that of ectopic hoxb2 expression, including ectopic krx20 (but not valentino) expression in the eye, and fusion of cartilages from pharyngeal arches 1 and 2. The results suggest that hoxb2 plays an important role in the patterning of hindbrain and pharyngeal arches in the zebrafish.

Exon skipping of AGAMOUS homolog PrseAG in developing double flowers of Prunus lannesiana (Rosaceae).

PubMed

Liu, Zhixiong; Zhang, Dandan; Liu, Di; Li, Fenglan; Lu, Hai

2013-02-01

KEY MESSAGE : Two transcript isoforms of AGAMOUS homologs, from single and double flower Prunus lannesiana, respectively, showed different functions. The Arabidopsis floral homeotic C function gene AGAMOUS (AG) confers stamen and carpel identity. Loss of AG function results in homeotic conversions of stamens into petals and formation of double flowers. In order to present a molecular dissection of a double-flower cultivar in Prunus lannesiana (Rosaceae), we isolated and identified a single-copy gene, AG homolog from two genetically cognate P. lannesiana bearing single and double flowers, respectively. Sequence analysis revealed that the AG homolog, prseag-1, from double flowers showed a 170-bp exon skipping as compared to PrseAG (Prunus serrulata AGAMOUS) from the single flowers. Genomic DNA sequence revealed that abnormal splicing resulted in mutant prseag-1 protein with the C-terminal AG motifs I and II deletions. In addition, protein sequence alignment and phylogenetic analyses revealed that the PrseAG was grouped into the euAG lineage. A semi-quantitative PCR analysis showed that the expression of PrseAG was restricted to reproductive organs of stamens and carpels in single flowers of P. lannesiana 'speciosa', while the prseag-1 mRNA was highly transcribed throughout the petals, stamens, and carpels in double flowers from 'Albo-rosea'. The transgenic Arabidopsis containing 35S::PrseAG displayed extremely early flowering, bigger stamens and carpels and homeotic conversion of petals into staminoid organs, but ectopic expression of prseag-1 could not mimic the phenotypic ectopic expression of PrseAG in Arabidopsis. In general, this study provides evidences to show that double flower 'Albo-rosea' is a putative C functional ag mutant in P. lannesiana.

MarvelD3 regulates the c-Jun N-terminal kinase pathway during eye development in Xenopus

PubMed Central

Vacca, Barbara; Sanchez-Heras, Elena; Steed, Emily; Balda, Maria S.; Ohnuma, Shin-Ichi; Sasai, Noriaki; Mayor, Roberto

2016-01-01

ABSTRACT Ocular morphogenesis requires several signalling pathways controlling the expression of transcription factors and cell-cycle regulators. However, despite a well-known mechanism, the dialogue between those signals and factors remains to be unveiled. Here, we identify a requirement for MarvelD3, a tight junction transmembrane protein, in eye morphogenesis in Xenopus. MarvelD3 depletion led to an abnormally pigmented eye or even an eye-less phenotype, which was rescued by ectopic MarvelD3 expression. Altering MarvelD3 expression led to deregulated expression of cell-cycle regulators and transcription factors required for eye development. The eye phenotype was rescued by increased c-Jun terminal Kinase activation. Thus, MarvelD3 links tight junctions and modulation of the JNK pathway to eye morphogenesis. PMID:27870636

The C. elegans che-1 gene encodes a zinc finger transcription factor required for specification of the ASE chemosensory neurons.

PubMed

Uchida, Okiko; Nakano, Hiroyuki; Koga, Makoto; Ohshima, Yasumi

2003-04-01

Chemotaxis to water-soluble chemicals such as NaCl is an important behavior of C. elegans when seeking food. ASE chemosensory neurons have a major role in this behavior. We show that che-1, defined by chemotaxis defects, encodes a zinc-finger protein similar to the GLASS transcription factor required for photoreceptor cell differentiation in Drosophila, and that che-1 is essential for specification and function of ASE neurons. Expression of a che-1::gfp fusion construct was predominant in ASE. In che-1 mutants, expression of genes characterizing ASE such as seven-transmembrane receptors, guanylate cyclases and a cyclic-nucleotide gated channel is lost. Ectopic expression of che-1 cDNA induced expression of ASE-specific marker genes, a dye-filling defect in neurons other than ASE and dauer formation.

High-dose bone morphogenetic protein-induced ectopic abdomen bone growth.

PubMed

Deutsch, Harel

2010-02-01

Infuse [bone morphogenetic protein (BMP)] is increasingly used in spinal fusion surgery. The authors report a rare complication of BMP use. This is a case report. A 55-year-old male underwent a thoracic T8 to the pelvis fusion for degenerative lumbar disc disease and pseudarthrosis at another institution. The procedure involved an anterior and posterior approach with the use of multiple units of BMP. The patient presented to our institution with complaints of weight loss, pain, tenderness, and increasing solid growth in the left lower quadrant several months after his surgery. A computed tomography revealed ectopic bone growth in the retroperitoneal area and pelvis contiguous to the anterior lumbar exposure. The anterior wound was re-explored, and a large sheet of ectopic bone was removed from the retroperitoneal space. We report a rare case of extraspinal ectopic bone growth because of the use of multiple packages of BMP. Copyright (c) 2010 Elsevier Inc. All rights reserved.

Status epilepticus increases mature granule cells in the molecular layer of the dentate gyrus in rats★

PubMed Central

Liang, Zhaoliang; Gao, Fei; Wang, Fajun; Wang, Xiaochen; Song, Xinyu; Liu, Kejing; Zhan, Ren-Zhi

2013-01-01

Enhanced neurogenesis in the dentate gyrus of the hippocampus following seizure activity, especially status epilepticus, is associated with ectopic residence and aberrant integration of newborn granule cells. Hilar ectopic granule cells may be detrimental to the stability of dentate circuitry by means of their electrophysiological properties and synaptic connectivity. We hypothesized that status epilepticus also increases ectopic granule cells in the molecular layer. Status epilepticus was induced in male Sprague-Dawley rats by intraperitoneal injection of pilocarpine. Immunostaining showed that many doublecortin-positive cells were present in the molecular layer and the hilus 7 days after the induction of status epilepticus. At least 10 weeks after status epilepticus, the estimated number of cells positive for both prospero homeobox protein 1 and neuron-specific nuclear protein in the hilus was significantly increased. A similar trend was also found in the molecular layer. These findings indicate that status epilepticus can increase the numbers of mature and ectopic newborn granule cells in the molecular layer. PMID:25206705

Zebrafish enpp1 mutants exhibit pathological mineralization, mimicking features of generalized arterial calcification of infancy (GACI) and pseudoxanthoma elasticum (PXE).

PubMed

Apschner, Alexander; Huitema, Leonie F A; Ponsioen, Bas; Peterson-Maduro, Josi; Schulte-Merker, Stefan

2014-07-01

In recent years it has become clear that, mechanistically, biomineralization is a process that has to be actively inhibited as a default state. This inhibition must be released in a rigidly controlled manner in order for mineralization to occur in skeletal elements and teeth. A central aspect of this concept is the tightly controlled balance between phosphate, a constituent of the biomineral hydroxyapatite, and pyrophosphate, a physiochemical inhibitor of mineralization. Here, we provide a detailed analysis of a zebrafish mutant, dragonfish (dgf), which is mutant for ectonucleoside pyrophosphatase/phosphodiesterase 1 (Enpp1), a protein that is crucial for supplying extracellular pyrophosphate. Generalized arterial calcification of infancy (GACI) is a fatal human disease, and the majority of cases are thought to be caused by mutations in ENPP1. Furthermore, some cases of pseudoxanthoma elasticum (PXE) have recently been linked to ENPP1. Similar to humans, we show here that zebrafish enpp1 mutants can develop ectopic calcifications in a variety of soft tissues - most notably in the skin, cartilage elements, the heart, intracranial space and the notochord sheet. Using transgenic reporter lines, we demonstrate that ectopic mineralizations in these tissues occur independently of the expression of typical osteoblast or cartilage markers. Intriguingly, we detect cells expressing the osteoclast markers Trap and CathepsinK at sites of ectopic calcification at time points when osteoclasts are not yet present in wild-type siblings. Treatment with the bisphosphonate etidronate rescues aspects of the dgf phenotype, and we detected deregulated expression of genes that are involved in phosphate homeostasis and mineralization, such as fgf23, npt2a, entpd5 and spp1 (also known as osteopontin). Employing a UAS-GalFF approach, we show that forced expression of enpp1 in blood vessels or the floorplate of mutant embryos is sufficient to rescue the notochord mineralization phenotype. This indicates that enpp1 can exert its function in tissues that are remote from its site of expression. © 2014. Published by The Company of Biologists Ltd.

The Ectopic Expression of the Wheat Puroindoline Genes Increase Germ Size and Seed Oil Content in Transgenic Corn

PubMed Central

Zhang, Jinrui; Martin, John M.; Beecher, Brian; Lu, Chaofu; Hannah, L. Curtis; Wall, Michael L.; Altosaar, Illimar; Giroux, Michael J.

2014-01-01

Plant oil content and composition improvement is a major goal of plant breeding and biotechnology. The Puroindoline a and b (PINA and PINB) proteins together control whether wheat seeds are soft or hard textured and share a similar structure to that of plant non-specific lipid-transfer proteins. Here we transformed corn (Zea mays L.) with the wheat (Triticum aestivum L.) puroindoline genes (Pina and Pinb) to assess their effects upon seed oil content and quality. Pina and Pinb coding sequences were introduced into corn under the control of a corn Ubiquitin promoter. Three Pina/Pinb expression positive transgenic events were evaluated over two growing seasons. The results showed that Pin expression increased germ size significantly without negatively impacting seed size. Germ yield increased 33.8% while total seed oil content was increased by 25.23%. Seed oil content increases were primarily the result of increased germ size. This work indicates that higher oil content corn hybrids having increased food or feed value could be produced via puroindoline expression. PMID:20725765

The role of Sox6 in zebrafish muscle fiber type specification.

PubMed

Jackson, Harriet E; Ono, Yosuke; Wang, Xingang; Elworthy, Stone; Cunliffe, Vincent T; Ingham, Philip W

2015-01-01

The transcription factor Sox6 has been implicated in regulating muscle fiber type-specific gene expression in mammals. In zebrafish, loss of function of the transcription factor Prdm1a results in a slow to fast-twitch fiber type transformation presaged by ectopic expression of sox6 in slow-twitch progenitors. Morpholino-mediated Sox6 knockdown can suppress this transformation but causes ectopic expression of only one of three slow-twitch specific genes assayed. Here, we use gain and loss of function analysis to analyse further the role of Sox6 in zebrafish muscle fiber type specification. The GAL4 binary misexpression system was used to express Sox6 ectopically in zebrafish embryos. Cis-regulatory elements were characterized using transgenic fish. Zinc finger nuclease mediated targeted mutagenesis was used to analyse the effects of loss of Sox6 function in embryonic, larval and adult zebrafish. Zebrafish transgenic for the GCaMP3 Calcium reporter were used to assay Ca2+ transients in wild-type and mutant muscle fibres. Ectopic Sox6 expression is sufficient to downregulate slow-twitch specific gene expression in zebrafish embryos. Cis-regulatory elements upstream of the slow myosin heavy chain 1 (smyhc1) and slow troponin c (tnnc1b) genes contain putative Sox6 binding sites required for repression of the former but not the latter. Embryos homozygous for sox6 null alleles expressed tnnc1b throughout the fast-twitch muscle whereas other slow-specific muscle genes, including smyhc1, were expressed ectopically in only a subset of fast-twitch fibers. Ca2+ transients in sox6 mutant fast-twitch fibers were intermediate in their speed and amplitude between those of wild-type slow- and fast-twitch fibers. sox6 homozygotes survived to adulthood and exhibited continued misexpression of tnnc1b as well as smaller slow-twitch fibers. They also exhibited a striking curvature of the spine. The Sox6 transcription factor is a key regulator of fast-twitch muscle fiber differentiation in the zebrafish, a role similar to that ascribed to its murine ortholog.

Up-Regulation of MiR-300 Promotes Proliferation and Invasion of Osteosarcoma by Targeting BRD7

PubMed Central

Xue, Zhen; Zhao, Jindong; Niu, Liyuan; An, Gang; Guo, Yashan; Ni, Linying

2015-01-01

Increasing reports suggest that deregulated microRNAs (miRNAs) might provide novel therapeutic targets for cancers. However, the expression and function of miR-300 in osteosarcoma is still unknown. In our study, we found that the expression of miR-300 was up-regulated in osteosarcoma tissues and cells compared with paired adjacent non-tumor bone tissues and osteoblastic cells using RT-qPCR. The enforced expression of miR-300 could promote cell proliferation, invasion and epithelial-mesenchymal transition (EMT). Moreover, we identified that bromodomain-containing protein 7 (BRD7), a new tumor suppressor gene, was a direct target of miR-300. Ectopic expression of BRD7 could significantly inhibit miR-300-promoted proliferation, invasion and EMT. Therefore, our results identify an important role for miR-300 in osteosarcoma through regulating BRD7 expression. PMID:26010572

Up-Regulation of MiR-300 Promotes Proliferation and Invasion of Osteosarcoma by Targeting BRD7.

PubMed

Xue, Zhen; Zhao, Jindong; Niu, Liyuan; An, Gang; Guo, Yashan; Ni, Linying

2015-01-01

Increasing reports suggest that deregulated microRNAs (miRNAs) might provide novel therapeutic targets for cancers. However, the expression and function of miR-300 in osteosarcoma is still unknown. In our study, we found that the expression of miR-300 was up-regulated in osteosarcoma tissues and cells compared with paired adjacent non-tumor bone tissues and osteoblastic cells using RT-qPCR. The enforced expression of miR-300 could promote cell proliferation, invasion and epithelial-mesenchymal transition (EMT). Moreover, we identified that bromodomain-containing protein 7 (BRD7), a new tumor suppressor gene, was a direct target of miR-300. Ectopic expression of BRD7 could significantly inhibit miR-300-promoted proliferation, invasion and EMT. Therefore, our results identify an important role for miR-300 in osteosarcoma through regulating BRD7 expression.

EBF proteins participate in transcriptional regulation of Xenopus muscle development.

PubMed

Green, Yangsook Song; Vetter, Monica L

2011-10-01

EBF proteins have diverse functions in the development of multiple lineages, including neurons, B cells and adipocytes. During Drosophila muscle development EBF proteins are expressed in muscle progenitors and are required for muscle cell differentiation, but there is no known function of EBF proteins in vertebrate muscle development. In this study, we examine the expression of ebf genes in Xenopus muscle tissue and show that EBF activity is necessary for aspects of Xenopus skeletal muscle development, including somite organization, migration of hypaxial muscle anlagen toward the ventral abdomen, and development of jaw muscle. From a microarray screen, we have identified multiple candidate targets of EBF activity with known roles in muscle development. The candidate targets we have verified are MYOD, MYF5, M-Cadherin and SEB-4. In vivo overexpression of the ebf2 and ebf3 genes leads to ectopic expression of these candidate targets, and knockdown of EBF activity causes downregulation of the endogenous expression of the candidate targets. Furthermore, we found that MYOD and MYF5 are likely to be direct targets. Finally we show that MYOD can upregulate the expression of ebf genes, indicating the presence of a positive feedback loop between EBF and MYOD that we find to be important for maintenance of MYOD expression in Xenopus. These results suggest that EBF activity is important for both stabilizing commitment and driving aspects of differentiation in Xenopus muscle cells. Copyright © 2010 Elsevier Inc. All rights reserved.

Seropositivity for the human heat shock protein (Hsp)60 accompanying seropositivity for Chlamydia trachomatis is less prevalent among tubal ectopic pregnancy cases than individuals with normal reproductive history.

PubMed

Ozyurek, Eser S; Karacan, Tolga; Ozdalgicoglu, Cenk; Yilmaz, Salih; Isik, Salman; San, Mevlide; Kaya, Erdal

2018-04-01

To investigate the role of anti-human heat shock protein 60 (hHsp60) antibody positivity in the pathogenesis of ectopic pregnancy, following Chlamydia trachomatis (CT) infection. In a case-control study, serological tests for anti-hHsp60 were performed in ectopic pregnancies (study group) and parturients with normal reproductive histories (control group). All participants in both groups were CT IgG(+). hHsp60 IgG(+) prevalences were compared between the two groups, by semiquantitative ELISA. Data were evaluated using nonparametric and parametric tests and multivariable regression. After an initial pilot study, two groups were formed: 63 ectopic gestations (study group) and 95 normal parturients (control group), all CT IgG(+). Blood samples from all cases were tested for anti-hHsp60 IgG. Age, gravidity, and practising contraception were higher in the control group, while a history of pelvic infections were more common in the study group. Hsp60 IgG(+) was found to be significantly higher in the control group (63/95, 66.3%) compared to study group (30/63, 47.6%). Regression analysis revealed anti-hHsp60 positivity was an independent factor delineating the two groups. Immunity to hHsp60 is less common in CT IgG(+) ectopic pregnancies than CT IgG(+) fertile subjects without a history of ectopic pregnancies. Hence, our findings suggest that hHsp60 seropositivity may decrease the probability of an ectopic gestation in subjects with previous CT infections. Copyright © 2018 Elsevier B.V. All rights reserved.

Epigenetic identification of ZNF545 as a functional tumor suppressor in multiple myeloma via activation of p53 signaling pathway

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fan, Yu; Zhan, Qian; Xu, Hongying

The KRAB–zinc-finger protein ZNF545 was recently identified as a potential suppressor gene in several tumors. However, the regulatory mechanisms of ZNF545 in tumorigenesis remain unclear. In this study, we investigated the expression and roles of ZNF545 in multiple myeloma (MM). ZNF545 was frequently downregulated in MM tissues compared with non-tumor bone marrow tissues. ZNF545 expression was silenced by promoter methylation in MM cell lines, and could be restored by demethylation treatment. ZNF545 methylation was detected in 28.3% of MM tissues, compared with 4.3% of normal bone marrow tissues. ZNF545 transcriptionally activated the p53 signaling pathway but had no effect onmore » Akt in MM, whereas ectopic expression of ZNF545 in silenced cells suppressed their proliferation and induced apoptosis. We therefore identified ZNF545 as a novel tumor suppressor inhibiting tumor growth through activation of the p53 pathway in MM. Moreover, tumor-specific methylation of ZNF545 may represent an epigenetic biomarker for MM diagnosis, and a potential target for specific therapy. -- Highlights: •Downregulated ZNF545 in MM tissues and cell lines and ectopic expression of ZNF545 suppresses tumor growth. •Tumor-specific methylation of ZNF545 represents an epigenetic biomarker for MM diagnosis, and a potential target for specific therapy. •ZNF545 exerts its tumor suppressive effects via transcriptional activating p53 pathway.« less

A Hox Gene, Antennapedia, Regulates Expression of Multiple Major Silk Protein Genes in the Silkworm Bombyx mori*

PubMed Central

Tsubota, Takuya; Tomita, Shuichiro; Uchino, Keiro; Kimoto, Mai; Takiya, Shigeharu; Kajiwara, Hideyuki; Yamazaki, Toshimasa; Sezutsu, Hideki

2016-01-01

Hox genes play a pivotal role in the determination of anteroposterior axis specificity during bilaterian animal development. They do so by acting as a master control and regulating the expression of genes important for development. Recently, however, we showed that Hox genes can also function in terminally differentiated tissue of the lepidopteran Bombyx mori. In this species, Antennapedia (Antp) regulates expression of sericin-1, a major silk protein gene, in the silk gland. Here, we investigated whether Antp can regulate expression of multiple genes in this tissue. By means of proteomic, RT-PCR, and in situ hybridization analyses, we demonstrate that misexpression of Antp in the posterior silk gland induced ectopic expression of major silk protein genes such as sericin-3, fhxh4, and fhxh5. These genes are normally expressed specifically in the middle silk gland as is Antp. Therefore, the evidence strongly suggests that Antp activates these silk protein genes in the middle silk gland. The putative sericin-1 activator complex (middle silk gland-intermolt-specific complex) can bind to the upstream regions of these genes, suggesting that Antp directly activates their expression. We also found that the pattern of gene expression was well conserved between B. mori and the wild species Bombyx mandarina, indicating that the gene regulation mechanism identified here is an evolutionarily conserved mechanism and not an artifact of the domestication of B. mori. We suggest that Hox genes have a role as a master control in terminally differentiated tissues, possibly acting as a primary regulator for a range of physiological processes. PMID:26814126

A Hox Gene, Antennapedia, Regulates Expression of Multiple Major Silk Protein Genes in the Silkworm Bombyx mori.

PubMed

Tsubota, Takuya; Tomita, Shuichiro; Uchino, Keiro; Kimoto, Mai; Takiya, Shigeharu; Kajiwara, Hideyuki; Yamazaki, Toshimasa; Sezutsu, Hideki

2016-03-25

Hoxgenes play a pivotal role in the determination of anteroposterior axis specificity during bilaterian animal development. They do so by acting as a master control and regulating the expression of genes important for development. Recently, however, we showed that Hoxgenes can also function in terminally differentiated tissue of the lepidopteranBombyx mori In this species,Antennapedia(Antp) regulates expression of sericin-1, a major silk protein gene, in the silk gland. Here, we investigated whether Antpcan regulate expression of multiple genes in this tissue. By means of proteomic, RT-PCR, and in situ hybridization analyses, we demonstrate that misexpression of Antpin the posterior silk gland induced ectopic expression of major silk protein genes such assericin-3,fhxh4, and fhxh5 These genes are normally expressed specifically in the middle silk gland as is Antp Therefore, the evidence strongly suggests that Antpactivates these silk protein genes in the middle silk gland. The putativesericin-1 activator complex (middle silk gland-intermolt-specific complex) can bind to the upstream regions of these genes, suggesting that Antpdirectly activates their expression. We also found that the pattern of gene expression was well conserved between B. moriand the wild species Bombyx mandarina, indicating that the gene regulation mechanism identified here is an evolutionarily conserved mechanism and not an artifact of the domestication of B. mori We suggest that Hoxgenes have a role as a master control in terminally differentiated tissues, possibly acting as a primary regulator for a range of physiological processes. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Expression and regulation of estrogen-converting enzymes in ectopic human endometrial tissue.

PubMed

Fechner, Sabine; Husen, Bettina; Thole, Hubert; Schmidt, Markus; Gashaw, Isabella; Kimmig, Rainer; Winterhager, Elke; Grümmer, Ruth

2007-10-01

To investigate the regulation of estrogen-converting enzymes in human ectopic endometrial tissue. Animal study. Academic medical center. Sixty female nude mice with implanted human endometrial tissue. Twenty-two premenopausal women undergoing endometrial biopsy or hysterectomy. Human endometrial tissue was implanted into the peritoneal cavity of nude mice, and the effect of therapeutic drugs on transcription of steroid receptors and estrogen-converting enzymes was analyzed. Transcript levels of steroid hormone receptors, 17beta-hydroxysteroid dehydrogenase type 1 and 2, aromatase, and steroid sulfatase as well as proliferation rate were analyzed in the human ectopic endometrial tissue. Steroid receptors and estrogen-converting enzymes were expressed in the ectopic human endometrial fragments. Application of medroxyprogesterone acetate, dydrogesterone, danazol, and the aromatase inhibitor finrozole significantly inhibited aromatase transcription. In addition, danazol caused a significant decrease in transcription of steroid sulfatase, and finrozole, of 17beta-hydroxysteroid dehydrogenase type 1 in parallel to a decrease in proliferation rate in the ectopic human endometrial tissue. Pharmacological regulation of transcription of estrogen-converting enzymes in human endometrium cultured in nude mice may help to develop new therapeutic concepts based on local regulation of estrogen metabolism in endometriosis.

Cytoplasm-predominant Pten associates with increased region-specific brain tyrosine hydroxylase and dopamine D2 receptors in mouse model with autistic traits.

PubMed

He, Xin; Thacker, Stetson; Romigh, Todd; Yu, Qi; Frazier, Thomas W; Eng, Charis

2015-01-01

Autism spectrum disorder (ASD) is a group of neurodevelopmental disorders characterized by impairment in social communication/interaction and inflexible/repetitive behavior. Several lines of evidence support genetic factors as a predominant cause of ASD. Among those autism susceptibility genes that have been identified, the PTEN tumor suppressor gene, initially identified as predisposing to Cowden heritable cancer syndrome, was found to be mutated in a subset of ASD patients with extreme macrocephaly. However, the ASD-relevant molecular mechanism mediating the effect of PTEN mutations remains elusive. We developed a Pten knock-in murine model to study the effects of Pten germline mutations, specifically altering subcellular localization, in ASD. Proteins were isolated from the hemispheres of the male littermates, and Western blots were performed to determine protein expression levels of tyrosine hydroxylase (TH). Immunohistochemical stains were carried out to validate the localization of TH and dopamine D2 receptors (D2R). PC12 cells ectopically expressing either wild-type or missense mutant PTEN were then compared for the differences in TH expression. Mice carrying Pten mutations have high TH and D2R in the striatum and prefrontal cortex. They also have increased phosphorylation of cAMP response element-binding protein (CREB) and TH. Mechanistically, PTEN downregulates TH production in PC12 cells via inhibiting the phosphoinositide 3-kinase (PI3K)/CREB signaling pathway, while PTEN reduces TH phosphorylation via suppressing MAPK pathway. Unlike wild-type PTEN but similar to the mouse knock-in mutant Pten, three naturally occurring missense mutations of PTEN that we previously identified in ASD patients, H93R, F241S, and D252G, were not able to suppress TH when overexpressed in PC12 cells. In addition, two other PTEN missense mutations, C124S (pan phosphatase dead) and G129E (lipid phosphatase dead), failed to suppress TH when ectopically expressed in PC12 cells. Our data reveal a non-canonical PTEN-TH pathway in the brain that may work as a core regulator of dopamine signaling, which when dysfunctional is pathogenic in ASD.

Gene conversion is strongly induced in human cells by double-strand breaks and is modulated by the expression of BCL-x(L)

NASA Technical Reports Server (NTRS)

Wiese, Claudia; Pierce, Andrew J.; Gauny, Stacey S.; Jasin, Maria; Kronenberg, Amy; Chatterjee, A. (Principal Investigator)

2002-01-01

Homology-directed repair (HDR) of DNA double-strand breaks (DSBs) contributes to the maintenance of genomic stability in rodent cells, and it has been assumed that HDR is of similar importance in DSB repair in human cells. However, some outcomes of homologous recombination can be deleterious, suggesting that factors exist to regulate HDR. We demonstrated previously that overexpression of BCL-2 or BCL-x(L) enhanced the frequency of X-ray-induced TK1 mutations, including loss of heterozygosity events presumed to arise by mitotic recombination. The present study was designed to test whether HDR is a prominent DSB repair pathway in human cells and to determine whether ectopic expression of BCL-x(L) affects HDR. Using TK6-neo cells, we find that a single DSB in an integrated HDR reporter stimulates gene conversion 40-50-fold, demonstrating efficient DSB repair by gene conversion in human cells. Significantly, DSB-induced gene conversion events are 3-4-fold more frequent in TK6 cells that stably overexpress the antiapoptotic protein BCL-X(L). Thus, HDR plays an important role in maintaining genomic integrity in human cells, and ectopic expression of BCL-x(L) enhances HDR of DSBs. This is the first study to highlight a function for BCL-x(L) in modulating DSB repair in human cells.

Interleukin-7-induced Stat-5 acts in synergy with Flt-3 signaling to stimulate expansion of hematopoietic progenitor cells.

PubMed

Åhsberg, Josefine; Tsapogas, Panagiotis; Qian, Hong; Zetterblad, Jenny; Zandi, Sasan; Månsson, Robert; Jönsson, Jan-Ingvar; Sigvardsson, Mikael

2010-11-19

The development of lymphoid cells from bone marrow progenitors is dictated by interplay between internal cues such as transcription factors and external signals like the cytokines Flt-3 ligand and Il-7. These proteins are both of large importance for normal lymphoid development; however, it is unclear if they act in direct synergy to expand a transient Il-7R(+)Flt-3(+) population or if the collaboration is created through sequential activities. We report here that Flt-3L and Il-7 synergistically stimulated the expansion of primary Il-7R(+)Flt-3(+) progenitor cells and a hematopoietic progenitor cell line ectopically expressing the receptors. The stimulation resulted in a reduced expression of pro-apoptotic genes and also mediated survival of primary progenitor cells in vitro. However, functional analysis of single cells suggested that the anti-apoptotic effect was additive indicating that the synergy observed mainly depends on stimulation of proliferation. Analysis of downstream signaling events suggested that although Il-7 induced Stat-5 phosphorylation, Flt-3L caused activation of the ERK and AKT signaling pathways. Flt-3L could also drive proliferation in synergy with ectopically expressed constitutively active Stat-5. This synergy could be inhibited with either receptor tyrosine kinase or MAPK inhibitors suggesting that Flt-3L and Il-7 act in synergy by activation of independent signaling pathways to expand early hematopoietic progenitors.

Long-Term Extensive Ectopic Hair Growth on the Spinal Cord of Mice from Transplanted Whisker Follicles.

PubMed

Cao, Wenluo; Li, Lingna; Mii, Sumiyuki; Amoh, Yasuyuki; Liu, Fang; Hoffman, Robert M

2015-01-01

We have previously demonstrated that hair follicles contain nestin-expressing pluripotent stem cells that can effect nerve and spinal cord repair upon transplantation. In the present study, isolated whisker follicles from nestin-driven green fluorescent protein (ND-GFP) mice were histocultured on Gelfoam for 3 weeks for the purpose of transplantation to the spinal cord to heal an induced injury. The hair shaft was cut off from Gelfoam-histocultured whisker follicles, and the remaining part of the whisker follicles containing GFP-nestin expressing pluripotent stem cells were transplanted into the injured spinal cord of nude mice, along with the Gelfoam. After 90 days, the mice were sacrificed and the spinal cord lesion was observed to have healed. ND-GFP expression was intense at the healed area of the spinal cord, as observed by fluorescence microscopy, demonstrating that the hair follicle stem cells were involved in healing the spinal cord. Unexpectedly, the transplanted whisker follicles sprouted out remarkably long hair shafts in the spinal cord during the 90 days after transplantation of Gelfoam whisker histocultures to the injured spine. The pigmented hair fibers, grown from the transplanted whisker histocultures, curved and enclosed the spinal cord. The unanticipated results demonstrate the great potential of hair growth after transplantation of Gelfoam hair follicle histocultures, even at an ectopic site.

Nanobodies and recombinant binders in cell biology.

PubMed

Helma, Jonas; Cardoso, M Cristina; Muyldermans, Serge; Leonhardt, Heinrich

2015-06-08

Antibodies are key reagents to investigate cellular processes. The development of recombinant antibodies and binders derived from natural protein scaffolds has expanded traditional applications, such as immunofluorescence, binding arrays, and immunoprecipitation. In addition, their small size and high stability in ectopic environments have enabled their use in all areas of cell research, including structural biology, advanced microscopy, and intracellular expression. Understanding these novel reagents as genetic modules that can be integrated into cellular pathways opens up a broad experimental spectrum to monitor and manipulate cellular processes. © 2015 Helma et al.

F-Box Protein FBX92 Affects Leaf Size in Arabidopsis thaliana

PubMed Central

Baute, Joke; Polyn, Stefanie; De Block, Jolien; Blomme, Jonas; Van Lijsebettens, Mieke

2017-01-01

F-box proteins are part of one of the largest families of regulatory proteins that play important roles in protein degradation. In plants, F-box proteins are functionally very diverse, and only a small subset has been characterized in detail. Here, we identified a novel F-box protein FBX92 as a repressor of leaf growth in Arabidopsis. Overexpression of AtFBX92 resulted in plants with smaller leaves than the wild type, whereas plants with reduced levels of AtFBX92 showed, in contrast, increased leaf growth by stimulating cell proliferation. Detailed cellular analysis suggested that AtFBX92 specifically affects the rate of cell division during early leaf development. This is supported by the increased expression levels of several cell cycle genes in plants with reduced AtFBX92 levels. Surprisingly, overexpression of the maize homologous gene ZmFBX92 in maize had no effect on plant growth, whereas ectopic expression in Arabidopsis increased leaf growth. Expression of a truncated form of AtFBX92 showed that the contrasting effects of ZmFBX92 and AtFBX92 gain of function in Arabidopsis are due to the absence of the F-box-associated domain in the ZmFBX92 gene. Our work reveals an additional player in the complex network that determines leaf size and lays the foundation for identifying putative substrates. PMID:28340173

RECK impedes DNA repair by inhibiting the erbB/JAB1/Rad51 signaling axis and enhances chemosensitivity of breast cancer cells

PubMed Central

Hong, Kun-Jing; Hsu, Ming-Chuan; Hung, Wen-Chun

2015-01-01

The reversion-inducing cysteine-rich protein with kazal motif (RECK) is an endogenous matrix metalloproteinase (MMP) inhibitor and a tumor suppressor. Its expression is dramatically down-regulated in human cancers. Our recent results suggest a novel MMP-independent anti-cancer activity of RECK by inhibiting the erbB signaling. Activation of the erbB signaling is associated with chemotherapeutic resistance, however, whether RECK could modulate drug sensitivity is still unknown. Here we demonstrated that expression of RECK induced the activation of ATM and ATR pathways, and the formation of γ-H2AX foci in breast cancer cells. RECK inhibited the erbB signaling and attenuated the expression of the downstream molecules Jun activation domain-binding protein 1 (JAB1) and the DNA repair protein RAD51 to impede DNA repair and to increase drug sensitivity. Treatment of epidermal growth factor or over-expression of HER-2 effectively reversed the inhibitory effect of RECK. In addition, ectopic expression of JAB1 counteracted RECK-induced RAD51 reduction and drug sensitization. Our results elucidate a novel function of RECK to modulate DNA damage response and drug resistance by inhibiting the erbB/Jab1/RAD51 signaling axis. Restoration of RECK expression in breast cancer cells may increase sensitivity to chemotherapeutic agents. PMID:26396917

High Expression of High-Mobility Group Box 1 in Menstrual Blood: Implications for Endometriosis.

PubMed

Shimizu, Keiko; Kamada, Yasuhiko; Sakamoto, Ai; Matsuda, Miwa; Nakatsuka, Mikiya; Hiramatsu, Yuji

2017-11-01

Endometriosis is a benign gynecologic disease characterized by the presence of ectopic endometrium and associated with inflammation and immune abnormalities. However, the molecular basis for endometriosis is not well understood. To address this issue, the present study examined the expression of high-mobility group box (HMGB) 1 in menstrual blood to investigate its role in the ectopic growth of human endometriotic stromal cells (ESCs). A total of 139 patients were enrolled in this study; 84 had endometriosis and 55 were nonendometriotic gynecological patients (control). The HMGB1 levels in various fluids were measured by enzyme-linked immunosorbent assay. Expression of receptor for advanced glycation end products (RAGE) in eutopic and ectopic endometrium was assessed by immunohistochemistry, and RAGE and vascular endothelial growth factor ( VEGF) messenger RNA expression in HMGB1- and lipopolysaccharide (LPS)-treated ESCs was evaluated by real-time polymerase chain reaction. The HMGB1 concentration was higher in menstrual blood than in serum or peritoneal fluid ( P < .001 for both). RAGE was expressed in both normal and ectopic endometrium. Administration of 1000 ng/mL HMGB1 or coadministration of 100 ng/mL HMGB1 and 100 ng/mL LPS induced VEGF production in ESCs relative to the control ( P < .05). These results suggest that menstrual fluid has naturally high levels of HMGB1 and may promote endometriosis following retrograde menstruation when complexed with other factors such as LPS by inducing inflammation and angiogenesis.

Cereal cystatins delay sprouting and nutrient loss in tubers of potato, Solanum tuberosum.

PubMed

Munger, Aurélie; Simon, Marie-Aube; Khalf, Moustafa; Goulet, Marie-Claire; Michaud, Dominique

2015-12-21

Recent studies have reported agronomically useful ectopic effects for recombinant protease inhibitors expressed in leaves of transgenic plants, including improved tolerance to abiotic stress conditions and partial resistance to necrotrophic pathogens. Here we assessed the effects of these proteins on the post-dormancy sprouting of storage organs, using as a model potato tubers expressing cysteine protease inhibitors of the cystatin protein superfamily. Sprout emergence and distribution, soluble proteins, starch and soluble sugars were monitored in tubers of cereal cystatin-expressing clones stored for several months at 4 °C. Cystatin expression had a strong repressing effect on sprout growth, associated with an apparent loss of apical dominance and an increased number of small buds at the skin surface. Soluble protein content remained high for up to 48 weeks in cystatin-expressing tubers compared to control (untransformed) tubers, likely explained by a significant stabilization of the major storage protein patatin, decreased hydrolysis of the endogenous protease inhibitor multicystatin and low cystatin-sensitive cysteine protease activity in tuber tissue. Starch content decreased after several months in cystatin-expressing tubers but remained higher than in control tubers, unlike sucrose showing a slower accumulation in the transgenics. Plantlet emergence, storage protein processing and height of growing plants showed similar time-course patterns for control and transgenic tubers, except for a systematic delay of 2 or 3 d in the latter group likely due to limited sprout size at sowing. Our data point overall to the onset of metabolic interference effects for cereal cystatins in sprouting potato tubers. They suggest, in practice, the potential of endogenous cysteine proteases as relevant targets for the development of potato varieties with longer storage capabilities.

CtBP impedes JNK- and Upd/STAT-driven cell fate misspecifications in regenerating Drosophila imaginal discs

PubMed Central

Worley, Melanie I; Alexander, Larissa A

2018-01-01

Regeneration following tissue damage often necessitates a mechanism for cellular re-programming, so that surviving cells can give rise to all cell types originally found in the damaged tissue. This process, if unchecked, can also generate cell types that are inappropriate for a given location. We conducted a screen for genes that negatively regulate the frequency of notum-to-wing transformations following genetic ablation and regeneration of the wing pouch, from which we identified mutations in the transcriptional co-repressor C-terminal Binding Protein (CtBP). When CtBP function is reduced, ablation of the pouch can activate the JNK/AP-1 and JAK/STAT pathways in the notum to destabilize cell fates. Ectopic expression of Wingless and Dilp8 precede the formation of the ectopic pouch, which is subsequently generated by recruitment of both anterior and posterior cells near the compartment boundary. Thus, CtBP stabilizes cell fates following damage by opposing the destabilizing effects of the JNK/AP-1 and JAK/STAT pathways. PMID:29372681

Transforming Growth Factor-β Promotes Liver Tumorigenesis in Mice via Up-regulation of Snail.

PubMed

Moon, Hyuk; Ju, Hye-Lim; Chung, Sook In; Cho, Kyung Joo; Eun, Jung Woo; Nam, Suk Woo; Han, Kwang-Hyub; Calvisi, Diego F; Ro, Simon Weonsang

2017-11-01

Transforming growth factor beta (TGF-β) suppresses early stages of tumorigenesis, but also contributes to migration and metastasis of cancer cells. A large number of human tumors contain mutations that inactivate its receptors, or downstream proteins such as Smad transcription factors, indicating that the TGF-β signaling pathway prevents tumor growth. We investigated the effects of TGF-β inhibition on liver tumorigenesis in mice. C57BL/6 mice received hydrodynamic tail-vein injections of transposons encoding HRAS G12V and a short hairpin RNA (shRNA) to down-regulate p53, or those encoding HRAS G12V and MYC, or those encoding HRAS G12V and TAZ S89A , to induce liver tumor formation; mice were also given injections of transposons encoding SMAD7 or shRNA against SMAD2, SMAD3, SMAD4, or SNAI1 (Snail), with or without ectopic expression of Snail. Survival times were compared, and livers were weighted and examined for tumors. Liver tumor tissues were analyzed by quantitative reverse-transcription PCR, RNA sequencing, immunoblots, and immunohistochemistry. We analyzed gene expression levels in human hepatocellular carcinoma samples deposited in The Cancer Genome Atlas. A cell proliferation assay was performed using human liver cancer cell lines (HepG2 and Huh7) stably expressing Snail or shRNA against Snail. TGF-β inhibition via overexpression of SMAD7 (or knockdown of SMAD2, SMAD3, or SMAD4) consistently reduced formation and growth of liver tumors in mice that expressed activated RAS plus shRNA against p53, or in mice that expressed activated RAS and TAZ. TGF-β signaling activated transcription of the Snail gene in liver tumors induced by HRAS G12V and shRNA against p53, and by activated RAS and TAZ. Knockdown of Snail reduced liver tumor formation in both tumor models. Ectopic expression of Snail restored liver tumorigenesis suppressed by disruption of TGF-β signaling. In human hepatocellular carcinoma, Snail expression correlated with TGF-β activation. Ectopic expression of Snail increased cellular proliferation, whereas Snail knockdown led to reduced proliferation in human hepatocellular carcinoma cells. In analyses of transgenic mice, we found TGF-β signaling to be required for formation of liver tumors upon expression of activated RAS and shRNA down-regulating p53, and upon expression of activated RAS and TAZ. Snail is the TGF-β target that is required for hepatic tumorigenesis in these models. Copyright © 2017 AGA Institute. Published by Elsevier Inc. All rights reserved.

MicroRNA-218 inhibits the proliferation of human choriocarcinoma JEG-3 cell line by targeting Fbxw8

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shi, Dazun; Tan, Zhihui; Lu, Rong

2014-08-08

Highlights: • The miR-218 expression was decreased in choriocarcinoma cell lines. • The Fbxw8 protein expression was increased in choriocarcinoma cell lines. • We show that Fbxw8 is bona-fide target of miR-218 in JEG-3. • Ectopic miR-218 expression inhibits the proliferation of JEG-3 via Fbxw8. • Overexpression of miR-218 affected cyclin A and p27 expression via Fbxw8. - Abstract: MicroRNAs (miRNAs) are endogenous 19–25 nucleotide noncoding single-stranded RNAs that regulate gene expression by blocking the translation or decreasing the stability of mRNAs. In this study, we showed that miR-218 expression levels were decreased while Fbxw8 expression levels were increased inmore » human choriocarcinoma cell lines, and identified Fbxw8 as a novel direct target of miR-218. Overexpression of miR-218 inhibited cell growth arrest at G2/M phase, suppressed the protein levels of cyclin A and up-regulated the expression levels of p27 through decreasing the levels of Fbxw8. On the other hand, forced expression of Fbxw8 partly rescued the effect of miR-218 in the cells, attenuated cell proliferation decrease the percentage of cells at G2/M phase, induced cyclin A protein expression and suppressed the protein level of p27 through up-regulating the levels of Fbxw8. Taken together, these findings will shed light the role to mechanism of miR-218 in regulating JEG-3 cells proliferation via miR-218/Fbxw8 axis, and miR-218 may serve as a novel potential therapeutic target in human choriocarcinoma in the future.« less

Silencing of secretory clusterin sensitizes NSCLC cells to V-ATPase inhibitors by downregulating survivin.

PubMed

Kim, Young-Sun; Jin, Hyeon-Ok; Hong, Sung-Eun; Song, Jie-Young; Hwang, Chang-Sun; Park, In-Chul

2018-01-08

Secretory clusterin (sCLU) is a stress-associated protein that confers resistance to therapy when overexpressed. In this study, we observed that the V-ATPase inhibitors bafilomycin A1 and concanamycin A significantly stimulated sCLU protein expression. Knockdown of sCLU with siRNA sensitized non-small cell lung cancer (NSCLC) cells to bafilomycin A1, suggesting that sCLU expression renders cells resistant to V-ATPase inhibitors. The dual PI3K/AKT and mTOR inhibitor BEZ235 suppressed sCLU expression and enhanced cell sensitivity induced by bafilomycin A1. Notably, sCLU knockdown further decreased the expression of the survivin protein by bafilomycin A1, and the ectopic expression of survivin alleviated the cell sensitivity by bafilomycin A1 and sCLU depletion, suggesting that increased sensitivity to sCLU depletion in the cells with V-ATPase inhibitors is due, at least in part, to the down-regulation of survivin. Taken together, we demonstrated that the depletion of sCLU expression enhances the sensitivity of NSCLC cells to V-ATPase inhibitors by decreasing survivin expression. Inhibition of the PI3K/AKT/mTOR pathway enhances the sensitivity of NSCLC cells to V-ATPase inhibitors, leading to decreased sCLU and survivin expression. Thus, we suggest that a combination of PI3K/AKT/mTOR inhibitors with V-ATPase inhibitors might be an effective approach for NSCLC treatment. Copyright © 2017 Elsevier Inc. All rights reserved.

Patterning of leaf vein networks by convergent auxin transport pathways.

PubMed

Sawchuk, Megan G; Edgar, Alexander; Scarpella, Enrico

2013-01-01

The formation of leaf vein patterns has fascinated biologists for centuries. Transport of the plant signal auxin has long been implicated in vein patterning, but molecular details have remained unclear. Varied evidence suggests a central role for the plasma-membrane (PM)-localized PIN-FORMED1 (PIN1) intercellular auxin transporter of Arabidopsis thaliana in auxin-transport-dependent vein patterning. However, in contrast to the severe vein-pattern defects induced by auxin transport inhibitors, pin1 mutant leaves have only mild vein-pattern defects. These defects have been interpreted as evidence of redundancy between PIN1 and the other four PM-localized PIN proteins in vein patterning, redundancy that underlies many developmental processes. By contrast, we show here that vein patterning in the Arabidopsis leaf is controlled by two distinct and convergent auxin-transport pathways: intercellular auxin transport mediated by PM-localized PIN1 and intracellular auxin transport mediated by the evolutionarily older, endoplasmic-reticulum-localized PIN6, PIN8, and PIN5. PIN6 and PIN8 are expressed, as PIN1 and PIN5, at sites of vein formation. pin6 synthetically enhances pin1 vein-pattern defects, and pin8 quantitatively enhances pin1pin6 vein-pattern defects. Function of PIN6 is necessary, redundantly with that of PIN8, and sufficient to control auxin response levels, PIN1 expression, and vein network formation; and the vein pattern defects induced by ectopic PIN6 expression are mimicked by ectopic PIN8 expression. Finally, vein patterning functions of PIN6 and PIN8 are antagonized by PIN5 function. Our data define a new level of control of vein patterning, one with repercussions on other patterning processes in the plant, and suggest a mechanism to select cell files specialized for vascular function that predates evolution of PM-localized PIN proteins.

Zinc finger protein 598 inhibits cell survival by promoting UV-induced apoptosis.

PubMed

Yang, Qiaohong; Gupta, Romi

2018-01-19

UV is one of the major causes of DNA damage induced apoptosis. However, cancer cells adopt alternative mechanisms to evade UV-induced apoptosis. To identify factors that protect cancer cells from UV-induced apoptosis, we performed a genome wide short-hairpin RNA (shRNA) screen, which identified Zinc finger protein 598 (ZNF598) as a key regulator of UV-induced apoptosis. Here, we show that UV irradiation transcriptionally upregulates ZNF598 expression. Additionally, ZNF598 knockdown in cancer cells inhibited UV-induced apoptosis. In our study, we observe that ELK1 mRNA level as well as phosphorylated ELK1 levels was up regulated upon UV irradiation, which was necessary for UV irradiation induced upregulation of ZNF598. Cells expressing ELK1 shRNA were also resistant to UV-induced apoptosis, and phenocopy ZNF598 knockdown. Upon further investigation, we found that ZNF598 knockdown inhibits UV-induced apoptotic gene expression, which matches with decrease in percentage of annexin V positive cell. Similarly, ectopic expression of ZNF598 promoted apoptotic gene expression and also increased annexin V positive cells. Collectively, these results demonstrate that ZNF598 is a UV irradiation regulated gene and its loss results in resistance to UV-induced apoptosis.

The involvement of J-protein AtDjC17 in root development in Arabidopsis

PubMed Central

Petti, Carloalberto; Nair, Meera; DeBolt, Seth

2014-01-01

In a screen for root hair morphogenesis mutants in Arabidopsis thaliana L. we identified a T-DNA insertion within a type III J-protein AtDjC17 caused altered root hair development and reduced hair length. Root hairs were observed to develop from trichoblast and atrichoblast cell files in both Atdjc17 and 35S::AtDJC17. Localization of gene expression in the root using transgenic plants expressing proAtDjC17::GUS revealed constitutive expression in stele cells. No AtDJC17 expression was observed in epidermal, endodermal, or cortical layers. To explore the contrast between gene expression in the stele and epidermal phenotype, hand cut transverse sections of Atdjc17 roots were examined showing that the endodermal and cortical cell layers displayed increased anticlinal cell divisions. Aberrant cortical cell division in Atdjc17 is proposed as causal in ectopic root hair formation via the positional cue requirement that exists between cortical and epidermal cell in hair cell fate determination. Results indicate a requirement for AtDJC17 in position-dependent cell fate determination and illustrate an intriguing requirement for molecular co-chaperone activity during root development. PMID:25339971

Histone Methylation Restrains the Expression of Subtype-Specific Genes during Terminal Neuronal Differentiation in Caenorhabditis elegans

PubMed Central

Chiang, Victor; Chalfie, Martin

2013-01-01

Although epigenetic control of stem cell fate choice is well established, little is known about epigenetic regulation of terminal neuronal differentiation. We found that some differences among the subtypes of Caenorhabditis elegans VC neurons, particularly the expression of the transcription factor gene unc-4, require histone modification, most likely H3K9 methylation. An EGF signal from the vulva alleviated the epigenetic repression of unc-4 in vulval VC neurons but not the more distant nonvulval VC cells, which kept unc-4 silenced. Loss of the H3K9 methyltransferase MET-2 or H3K9me2/3 binding proteins HPL-2 and LIN-61 or a novel chromodomain protein CEC-3 caused ectopic unc-4 expression in all VC neurons. Downstream of the EGF signaling in vulval VC neurons, the transcription factor LIN-11 and histone demethylases removed the suppressive histone marks and derepressed unc-4. Behaviorally, expression of UNC-4 in all the VC neurons caused an imbalance in the egg-laying circuit. Thus, epigenetic mechanisms help establish subtype-specific gene expression, which are needed for optimal activity of a neural circuit. PMID:24348272

Claudin-1 induced sealing of blood-brain barrier tight junctions ameliorates chronic experimental autoimmune encephalomyelitis.

PubMed

Pfeiffer, Friederike; Schäfer, Julia; Lyck, Ruth; Makrides, Victoria; Brunner, Sarah; Schaeren-Wiemers, Nicole; Deutsch, Urban; Engelhardt, Britta

2011-11-01

In experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis (MS), loss of the blood-brain barrier (BBB) tight junction (TJ) protein claudin-3 correlates with immune cell infiltration into the CNS and BBB leakiness. Here we show that sealing BBB TJs by ectopic tetracycline-regulated expression of the TJ protein claudin-1 in Tie-2 tTA//TRE-claudin-1 double transgenic C57BL/6 mice had no influence on immune cell trafficking across the BBB during EAE and furthermore did not influence the onset and severity of the first clinical disease episode. However, expression of claudin-1 did significantly reduce BBB leakiness for both blood borne tracers and endogenous plasma proteins specifically around vessels expressing claudin-1. In addition, mice expressing claudin-1 exhibited a reduced disease burden during the chronic phase of EAE as compared to control littermates. Our study identifies BBB TJs as the critical structure regulating BBB permeability but not immune cell trafficking into CNS during EAE, and indicates BBB dysfunction is a potential key event contributing to disease burden in the chronic phase of EAE. Our observations suggest that stabilizing BBB barrier function by therapeutic targeting of TJs may be beneficial in treating MS, especially when anti-inflammatory treatments have failed.

Induction of Pancreatic Differentiation by Signals from Blood Vessels

NASA Astrophysics Data System (ADS)

Lammert, Eckhard; Cleaver, Ondine; Melton, Douglas

2001-10-01

Blood vessels supply developing organs with metabolic sustenance. Here, we demonstrate a role for blood vessels as a source of developmental signals during pancreatic organogenesis. In vitro experiments with embryonic mouse tissues demonstrate that blood vessel endothelium induces insulin expression in isolated endoderm. Removal of the dorsal aorta in Xenopus laevis embryos results in the failure of insulin expression in vivo. Furthermore, using transgenic mice, we show that ectopic vascularization in the posterior foregut leads to ectopic insulin expression and islet hyperplasia. These results indicate that vessels not only provide metabolic sustenance, but also provide inductive signals for organ development.

Ras/Mitogen-activated Protein Kinase (MAPK) Signaling Modulates Protein Stability and Cell Surface Expression of Scavenger Receptor SR-BI*

PubMed Central

Wood, Peta; Mulay, Vishwaroop; Darabi, Masoud; Chan, Karen Cecilia; Heeren, Joerg; Pol, Albert; Lambert, Gilles; Rye, Kerry-Anne; Enrich, Carlos; Grewal, Thomas

2011-01-01

The mitogen-activated protein kinase (MAPK) Erk1/2 has been implicated to modulate the activity of nuclear receptors, including peroxisome proliferator activator receptors (PPARs) and liver X receptor, to alter the ability of cells to export cholesterol. Here, we investigated if the Ras-Raf-Mek-Erk1/2 signaling cascade could affect reverse cholesterol transport via modulation of scavenger receptor class BI (SR-BI) levels. We demonstrate that in Chinese hamster ovary (CHO) and human embryonic kidney (HEK293) cells, Mek1/2 inhibition reduces PPARα-inducible SR-BI protein expression and activity, as judged by reduced efflux onto high density lipoprotein (HDL). Ectopic expression of constitutively active H-Ras and Mek1 increases SR-BI protein levels, which correlates with elevated PPARα Ser-21 phosphorylation and increased cholesterol efflux. In contrast, SR-BI levels are insensitive to Mek1/2 inhibitors in PPARα-depleted cells. Most strikingly, Mek1/2 inhibition promotes SR-BI degradation in SR-BI-overexpressing CHO cells and human HuH7 hepatocytes, which is associated with reduced uptake of radiolabeled and 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyane-labeled HDL. Loss of Mek1/2 kinase activity reduces SR-BI expression in the presence of bafilomycin, an inhibitor of lysosomal degradation, indicating down-regulation of SR-BI via proteasomal pathways. In conclusion, Mek1/2 inhibition enhances the PPARα-dependent degradation of SR-BI in hepatocytes. PMID:21525007

Periostin, a matrix specific protein, is associated with proliferation and invasion of pancreatic cancer.

PubMed

Ben, Qi-Wen; Jin, Xiao-Long; Liu, Jun; Cai, Xia; Yuan, Fei; Yuan, Yao-Zong

2011-03-01

Overexpression of periostin is present in various malignant tumors and correlates with disease progression. However, its clinicopathological significance in pancreatic cancer is currently not known. Expression of periostin was analyzed by RT-PCR and western blotting in pancreatic cancers and cell lines. Using immunohistochemistry, expression of periostin in pancreatic cancers was evaluated according to factors influencing overall survival with Kaplan-Meier analysis. Ectopic expression of periostin was used to examine the effects of periostin on proliferation and invasiveness of pancreatic cancer cells in vitro. There was no detectable periostin mRNA and protein expression in the 4 pancreatic cell lines. Expression of periostin was found to be up-regulated in pancreatic cancer compared to the adjacent tumor free (TF) tissues by western blotting. The positive ratio of periostin expression in the neoplastic stroma was significantly correlated with the depth of invasion (p=0.007) and lymph node metastasis (p=0.027). Survival analysis showed that stromal or epithelium expression of periostin was associated with poor survival (p=0.035, p=0.022, log-rank test, respectively). In vitro studies showed that periostin was able to promote proliferation and invasiveness of pancreatic cancer cells. These results suggest that periostin may be involved in the progression and invasion of pancreatic cancer.

Inflammatory and age-related pathologies in mice with ectopic expression of human PARP-1.

PubMed

Mangerich, Aswin; Herbach, Nadja; Hanf, Benjamin; Fischbach, Arthur; Popp, Oliver; Moreno-Villanueva, María; Bruns, Oliver T; Bürkle, Alexander

2010-06-01

Poly(ADP-ribose) polymerase-1 (PARP-1) is a sensor for DNA strand breaks and some unusual DNA structures and catalyzes poly(ADP-ribosyl)ation of nuclear proteins with NAD(+) serving as substrate. PARP-1 is involved in the regulation of genomic integrity, transcription, inflammation, and cell death. Due to its versatile role, PARP-1 is discussed both as a longevity factor and as an aging-promoting factor. Recently, we generated a mouse model with ectopic integration of full-length hPARP-1 [Mangerich, A., Scherthan, H., Diefenbach, J., Kloz, U., van der Hoeven, F., Beneke, S. and Bürkle, A., 2009. A caveat in mouse genetic engineering: ectopic gene targeting in ES cells by bidirectional extension of the homology arms of a gene replacement vector carrying human PARP-1. Transgenic Res. 18, 261-279]. Here, we show that hPARP-1 mice exhibit impaired survival rates accompanied by reduced hair growth and premature development of several inflammation and age-associated pathologies, such as adiposity, kyphosis, nephropathy, dermatitis, pneumonitis, cardiomyopathy, hepatitis, and anemia. Moreover, mutant male mice showed impaired glucose tolerance, yet without developing manifest diabetes. Overall tumor burden was comparable in wild-type and hPARP-1 mice, but tumor spectrum was shifted in mutant mice, showing lower incidence of sarcomas, but increased incidence of carcinomas. Furthermore, DNA repair was delayed in splenocytes of hPARP-1 mice, and gene expression of pro-inflammatory cytokines was dysregulated. Our results suggest that in hPARP-1 mice impaired DNA repair, accompanied by a continuous low-level increase in pro-inflammatory stimuli, causes development of chronic diseases leading to impaired survival. (c) 2010 Elsevier Ireland Ltd. All rights reserved.

Characterization of the synthesis and expression of the GTA-kinase from transformed and normal rodent cells.

PubMed

Kerr, M; Fischer, J E; Purushotham, K R; Gao, D; Nakagawa, Y; Maeda, N; Ghanta, V; Hiramoto, R; Chegini, N; Humphreys-Beher, M G

1994-08-02

The murine transformed cell line YC-8 and beta-adrenergic receptor agonist (isoproternol) treated rat and mouse parotid gland acinar cells ectopically express cell surface beta 1-4 galactosyltransferase during active proliferation. This activity is dependent upon the expression of the GTA-kinase (p58) in these cells. Using total RNA, cDNA clones for the protein coding region of the kinase were isolated by reverse transcriptase-PCR cloning. DNA sequence analysis failed to show sequence differences with the normal homolog from mouse cells although Southern blot analysis of YC-8, and a second cell line KI81, indicated changes in the restriction enzyme digestion profile relative to murine cell lines which do not express cell surface galactosyltransferase. The rat cDNA clone from isoproterenol-treated salivary glands showed a high degree of protein and nucleic acid sequence homology to the GTA-kinase from both murine and human sources. Northern blot analysis of YC-8 and a control cell line LSTRA revealed the synthesis of a major 3.0 kb mRNA from both cell lines plus the unique expression of a 4.5 kb mRNA in the YC-8 cells. Reverse transcriptase-PCR of LSTRA and YC-8 confirmed the increased steady state levels of the GTA-kinase mRNA in YC-8. In the mouse, induction of cell proliferation by isoproterenol resulted in a 50-fold increase in steady state mRNA levels for the kinase over the low level of expression in quiescent cells. Expression of the rat 3' untranslated region in rat parotid cells in vitro led to an increased rate of DNA synthesis, cell number an ectopic expression of cell surface galactosyltransferase in the sense orientation. Antisense expression or vector alone did not alter growth characteristics of acinar cells. A polyclonal antibody monospecific to a murine amino terminal peptide sequence revealed a uniform distribution of GTA-kinase over the cytoplasm of acinar and duct cells of control mouse parotid glands. However, upon growth stimulation, kinase was detected primarily in a perinuclear and nuclear immunostaining pattern. Western blot analysis confirmed a translocation from a cytoplasmic localization in both LSTRA and quiescent salivary cells to a membrane-associated localization in YC-8 and proliferating salivary cells.

The role of ZAP70 kinase in acute lymphoblastic leukemia infiltration into the central nervous system.

PubMed

Alsadeq, Ameera; Fedders, Henning; Vokuhl, Christian; Belau, Nele M; Zimmermann, Martin; Wirbelauer, Tim; Spielberg, Steffi; Vossen-Gajcy, Michaela; Cario, Gunnar; Schrappe, Martin; Schewe, Denis M

2017-02-01

Central nervous system infiltration and relapse are poorly understood in childhood acute lymphoblastic leukemia. We examined the role of zeta-chain-associated protein kinase 70 in preclinical models of central nervous system leukemia and performed correlative studies in patients. Zeta-chain-associated protein kinase 70 expression in acute lymphoblastic leukemia cells was modulated using short hairpin ribonucleic acid-mediated knockdown or ectopic expression. We show that zeta-chain-associated protein kinase 70 regulates CCR7/CXCR4 via activation of extracellular signal-regulated kinases. High expression of zeta-chain-associated protein kinase 70 in acute lymphoblastic leukemia cells resulted in a higher proportion of central nervous system leukemia in xenografts as compared to zeta-chain-associated protein kinase 70 low expressing counterparts. High zeta-chain-associated protein kinase 70 also enhanced the migration potential towards CCL19/CXCL12 gradients in vitro CCR7 blockade almost abrogated homing of acute lymphoblastic leukemia cells to the central nervous system in xenografts. In 130 B-cell precursor acute lymphoblastic leukemia and 117 T-cell acute lymphoblastic leukemia patients, zeta-chain-associated protein kinase 70 and CCR7/CXCR4 expression levels were significantly correlated. Zeta-chain-associated protein kinase 70 expression correlated with central nervous system disease in B-cell precursor acute lymphoblastic leukemia, and CCR7/CXCR4 correlated with central nervous system involvement in T-cell acute lymphoblastic leukemia patients. In multivariate analysis, zeta-chain-associated protein kinase 70 expression levels in the upper third and fourth quartiles were associated with central nervous system involvement in B-cell precursor acute lymphoblastic leukemia (odds ratio=7.48, 95% confidence interval, 2.06-27.17; odds ratio=6.86, 95% confidence interval, 1.86-25.26, respectively). CCR7 expression in the upper fourth quartile correlated with central nervous system positivity in T-cell acute lymphoblastic leukemia (odds ratio=11.00, 95% confidence interval, 2.00-60.62). We propose zeta-chain-associated protein kinase 70, CCR7 and CXCR4 as markers of central nervous system infiltration in acute lymphoblastic leukemia warranting prospective investigation. Copyright© Ferrata Storti Foundation.

Exploiting cancer’s phenotypic guise against itself: targeting ectopically expressed peptide G-protein coupled receptors for lung cancer therapy

PubMed Central

Khan, Mahjabin; Huang, Tao; Lin, Cheng-Yuan; Wu, Jiang; Fan, Bao-Min; Bian, Zhao-Xiang

2017-01-01

Lung cancer, claiming millions of lives annually, has the highest mortality rate worldwide. This advocates the development of novel cancer therapies that are highly toxic for cancer cells but negligibly toxic for healthy cells. One of the effective treatments is targeting overexpressed surface receptors of cancer cells with receptor-specific drugs. The receptors-in-focus in the current review are the G-protein coupled receptors (GPCRs), which are often overexpressed in various types of tumors. The peptide subfamily of GPCRs is the pivot of the current article owing to the high affinity and specificity to and of their cognate peptide ligands, and the proven efficacy of peptide-based therapeutics. The article summarizes various ectopically expressed peptide GPCRs in lung cancer, namely, Cholecystokinin-B/Gastrin receptor, the Bombesin receptor family, Bradykinin B1 and B2 receptors, Arginine vasopressin receptors 1a, 1b and 2, and the Somatostatin receptor type 2. The autocrine growth and pro-proliferative pathways they mediate, and the distinct tumor-inhibitory effects of somatostatin receptors are then discussed. The next section covers how these pathways may be influenced or ‘corrected’ through therapeutics (involving agonists and antagonists) targeting the overexpressed peptide GPCRs. The review proceeds on to Nano-scaled delivery platforms, which enclose chemotherapeutic agents and are decorated with peptide ligands on their external surface, as an effective means of targeting cancer cells. We conclude that targeting these overexpressed peptide GPCRs is potentially evolving as a highly promising form of lung cancer therapy. PMID:29262666

Suppression of Akt-mediated HDAC3 expression and CDK2 T39 phosphorylation by a bichalcone analog contributes to S phase retardation of cancer cells.

PubMed

Hung, Kuang-Chen; Lin, Meng-Liang; Hsu, Shih-Wei; Lee, Chuan-Chun; Huang, Ren-Yu; Wu, Tian-Shung; Chen, Shih-Shun

2018-06-15

Targeting cell cycle regulators has been a suggested mechanism for therapeutic cancer strategies. We report here that the bichalcone analog TSWU-CD4 induces S phase arrest of human cancer cells by inhibiting the formation of cyclin A-phospho (p)-cyclin-dependent kinase 2 (CDK2, threonine [Thr] 39) complexes, independent of mutant p53 expression. Ectopic expression of CDK2 (T39E), which mimics phosphorylation of the Thr 39 residue of CDK2, partially rescues the cells from TSWU-CD4-induced S phase arrest, whereas phosphorylation-deficient CDK2 (T39A) expression regulates cell growth with significant S phase arrest and enhances TSWU-CD4-triggered S phase arrest. Decreased histone deacetylase 3 (HDAC3) expression after TSWU-CD4 treatment was demonstrated, and TSWU-CD4 induced S phase arrest and inhibitory effects on cyclin A expression and CDK2 Thr 39 phosphorylation, while cyclin A-p-CDK2 (Thr 39) complex formation was suppressed by ectopic wild-type HDAC3 expression. The co-transfection of CDK2 (T39E) along with HDAC3 completely restored cyclin A expression, Thr 39-phosphorylated CDK2, cyclin A-p-CDK2 (Thr 39) complex formation, and the S phase population to normal levels. Protein kinase B (Akt) inactivation was required for TSWU-CD4-induced S phase cell cycle arrest, because constitutively active Akt1 blocks the induction of S phase arrest and the suppression of cyclin A and HDAC3 expression, CDK2 Thr 39 phosphorylation, and cyclin A-p-CDK2 (Thr 39) complex formation by TSWU-CD4. Taken together, our results indicate that TSWU-CD4 induces S phase arrest by inhibiting Akt-mediated HDAC3 expression and CDK2 Thr 39 phosphorylation to suppress the formation of cyclin A-p-CDK2 (Thr 39) complexes. Copyright © 2018 Elsevier B.V. All rights reserved.

MicroRNA-466 (miR-466) functions as a tumor suppressor and prognostic factor in colorectal cancer (CRC).

PubMed

Tong, Feng; Ying, Youhua; Pan, Haihua; Zhao, Wei; Li, Hongchen; Zhan, Xiaoli

2018-01-17

MicroRNAs (miRNAs) have an important role in the regulation of tumor development and metastasis. In this study, we investigated the clinical and prognostic value as well as biological function of miR-466 in colorectal cancer (CRC). Tumor and adjacent healthy tissues were obtained from 100 patients diagnosed with CRC. miR-466 expression was determined by quantitative reverse transcription polymerase chain reaction (qRT-PCR). mRNA and protein levels of cyclin D1, apoptosis regulator BAX (BAX), and matrix metalloproteinase-2 (MMP-2) were analyzed by qRT-PCR and Western blot, respectively, in SW-620 CRC cells transfected with miR-466 mimics or negative control miRNA. Effects of miR-466 on SW-620 cell proliferation, cell cycle and apoptosis, and invasion were investigated using CCK-8 assay, flow cytometry and Transwell assay, respectively. miR-466 expression was significantly downregulated in tumor tissues compared to matched adjacent non-tumor tissues. Low expression of miR-466 was significantly correlated with the tumor size, Tumor Node Metastasis stage, lymph node metastasis, and distant metastasis. The overall survival of CRC patients with low miR-466 expression was significantly shorter compared to high-miR-466 expression group (log-rank test: p = 0.0103). Multivariate analysis revealed that low miR-466 expression was associated with poor prognosis in CRC patients. The ectopic expression of miR-466 suppressed cell proliferation and migration/invasion, as well as induced G0/G1 arrest and apoptosis in SW-620 cells. Moreover, the ectopic expression of miR-466 decreased the expression of cyclin D1 and MMP-2, but increased BAX expression in SW-620 cells. In conclusion, our findings demonstrated that miR-466 functions as a suppressor miRNA in CRC and may be used as a prognostic factor in these patients.

Ectopic expression of protein kinase C-β sensitizes head and neck squamous cell carcinoma to diterpene esters.

PubMed

Adams, Ryan A; D'Souza, Marjorie M A; Pierce, Carly J; Korica, Natasa; Wallwork, Ben; Parsons, Peter G; Panizza, Benedict; Boyle, Glen M

2015-03-01

The objective of this study was to examine the effect of specific Protein kinase C (PKC) isoform re-expression in solid malignancies, particularly head and neck squamous cell carcinoma cell lines, and the impact this may have on treatment with known activators of PKC. The constitutive expression of PKC isoforms were determined in six head and neck squamous cell carcinoma (SCC) cell lines. Cytotoxicity of the prototypic phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA) and the novel diterpene ester PEP005 was established. Viral transduction to re-express PKCβ isoforms in two of these cell lines was performed, and its effect on the sensitivity to the compounds was quantified. Tongue and hypopharyngeal SCC cell lines were resistant to both TPA and PEP005, with the concentration required to inhibit growth by 50% (IC50) being >1,000 ng/ml. CAL-27 (tongue SCC) and FaDu (hypopharyngeal SCC) cell lines re-expressing PKCβI and -βII isoforms demonstrated IC50 of 1-5 ng/ml with TPA or PEP005. Re-expression of PKCβ in head and neck SCC cell lines leads to cells one thousand-times more sensitive to the cytotoxic effects of phorbol or diterpene esters in culture. This highlights the importance of the isoform in tumor progression and presents the potential benefit of these compounds in malignancies expressing the protein, and in combination therapy. Copyright© 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

Endogenous and ectopic expression of telomere regulating genes in chicken embryonic fibroblasts

DOE Office of Scientific and Technical Information (OSTI.GOV)

Michailidis, Georgios; Saretzki, Gabriele; Hall, Judith

In this study, we compared the endogenous expression of genes encoding telomere regulating proteins in cultured chicken embryonic fibroblasts (CEFs) and 10-day-old chicken embryos. CEFs maintained in vitro senesced and senescence was accompanied by reduced telomere length, telomerase activity, and expression of the chicken (c) TRF1 gene. There was no change in TRF2 gene expression although the major TRF2 transcript identified in 10-day-old chicken embryos encoded a truncated TRF2 protein (TRF2'), containing an N-terminal dimerisation domain but lacking a myb-related DNA binding domain and nuclear localisation signal. Senescence of the CEFs in vitro was associated with the loss of themore » TRF2' transcript, indicative of a novel function for the encoded protein. Senescence was also coupled with decreased expression of RAD51, but increased RAD52 expression. These data support that RAD51 independent recombination mechanisms do not function in vitro to maintain chicken telomeres. To attempt to rescue the CEFs from replicative senescence, we stably transfected passage 3 CEFs with the human telomerase reverse transcriptase (hTERT) catalytic subunit. While hTERT expression was detected in the stable transfectants neither telomerase activity nor the stabilisation of telomere length was observed, and the transfectant cells senesced at the same passage number as the untransfected cells. These data indicate that the human TERT is incompatible with the avian telomere maintenance apparatus and suggest the functioning of a species specific telomere system in the avian.« less

F-actin clustering and cell dysmotility induced by the pathological W148R missense mutation of filamin B at the actin-binding domain.

PubMed

Zhao, Yongtong; Shapiro, Sandor S; Eto, Masumi

2016-01-01

Filamin B (FLNB) is a dimeric actin-binding protein that orchestrates the reorganization of the actin cytoskeleton. Congenital mutations of FLNB at the actin-binding domain (ABD) are known to cause abnormalities of skeletal development, such as atelosteogenesis types I and III and Larsen's syndrome, although the underlying mechanisms are poorly understood. Here, using fluorescence microscopy, we characterized the reorganization of the actin cytoskeleton in cells expressing each of six pathological FLNB mutants that have been linked to skeletal abnormalities. The subfractionation assay showed a greater accumulation of the FLNB ABD mutants W148R and E227K than the wild-type protein to the cytoskeleton. Ectopic expression of FLNB-W148R and, to a lesser extent, FLNB-E227K induced prominent F-actin accumulations and the consequent rearrangement of focal adhesions, myosin II, and septin filaments and results in a delayed directional migration of the cells. The W148R protein-induced cytoskeletal rearrangement was partially attenuated by the inhibition of myosin II, p21-activated protein kinase, or Rho-associated protein kinase. The expression of a single-head ABD fragment with the mutations partially mimicked the rearrangement induced by the dimer. The F-actin clustering through the interaction with the mutant FLNB ABD may limit the cytoskeletal reorganization, preventing normal skeletal development. Copyright © 2016 the American Physiological Society.

Molecular Mechanisms Regulating Muscle Fiber Composition Under Microgravity

NASA Technical Reports Server (NTRS)

Rosenthal, Nadia A.

1999-01-01

The overall goal of this project is to reveal the molecular mechanisms underlying the selective and debilitating atrophy of specific skeletal muscle fiber types that accompanies sustained conditions of microgravity. Since little is currently known about the regulation of fiber-specific gene expression programs in mammalian muscle, elucidation of the basic mechanisms of fiber diversification is a necessary prerequisite to the generation of therapeutic strategies for attenuation of muscle atrophy on earth or in space. Vertebrate skeletal muscle development involves the fusion of undifferentiated mononucleated myoblasts to form multinucleated myofibers, with a concomitant activation of muscle-specific genes encoding proteins that form the force-generating contractile apparatus. The regulatory circuitry controlling skeletal muscle gene expression has been well studied in a number of vertebrate animal systems. The goal of this project has been to achieve a similar level of understanding of the mechanisms underlying the further specification of muscles into different fiber types, and the role played by innervation and physical activity in the maintenance and adaptation of different fiber phenotypes into adulthood. Our recent research on the genetic basis of fiber specificity has focused on the emergence of mature fiber types and have implicated a group of transcriptional regulatory proteins, known as E proteins, in the control of fiber specificity. The restriction of E proteins to selected muscle fiber types is an attractive hypothetical mechanism for the generation of muscle fiber-specific patterns of gene expression. To date our results support a model wherein different E proteins are selectively expressed in muscle cells to determine fiber-restricted gene expression. These studies are a first step to define the molecular mechanisms responsible for the shifts in fiber type under conditions of microgravity, and to determine the potential importance of E proteins as upstream targets for the effects of weightlessness. In the past year we have determined that the expression of E Proteins is restricted to specific fiber types by post-transcriptional mechanisms. By far, the most prevalent mechanism of cellular control for achieving post-transcriptional regulation of gene expression is selective proteolysis -through the ubiquitin -proteasome pathway. Steady-state levels of HEB message are similar in all fast and slow skeletal muscle fiber types, yet the protein is restricted to Type IIX fibers. HEB appears to be a nodal point for regulating fiber-specific transcription, as expression of the transcription factor is regulated at the post-transcriptional level. It is not clear at present whether the regulation is at the level of protein synthesis or degradation. We are now poised to evaluate the biological role of ubiquitination in fiber specific-gene expression by controlling the post-transcriptional expression of E Proteins. The use of metabolic labelling and pharmacological inhibitors of the ubiquitin pathway will be used to identify the mode of regulation of the Type IIX expression pattern. The potential role of specific kinases in effecting the restriction of HEB expression will be examined by using both inhibitors and activators. The results of these studies will provide the necessary information to evaluate the biological role of E proteins in controlling fiber type transitions, and in potentially attenuating the atrophic effects of microgravity conditions. We have also recently shown that ectopic expression of the HEB protein transactivates the Type IIX-specific skeletal a-actin reporter. The 218 bp skeletal a-actin promoter drives transgene expression solely in mature Type IIX fibers. A mouse also carrying the transgene MLCI/HEB (which ectopically expresses the E Protein HEB in Type IIB fibers) forces expression of the skeletal a-actin reporter gene in Type IIB fibers. We can now dissect the composition of this fiber-specific cis-element. The skeletal a-actin promoter is quite compact and has been extensively characterized in vitro for activity and binding factors. The single E box may act as a binding target of myogenic factor/HEB heterodimer to allow for IIX expression. The HEB transcription factor may recognize either the precise flanking sequences of the E Box, or perhaps interacting with other proteins bound nearby, and activating expression in Type IIX fibers. This E box will be both ablated, and alternatively, as ablation may well destroy any muscle-specific transcriptional activity, flanking sequences substituted with those surrounding the E box (El) of the myogenin promoter. Modification of fiber-specific transgene expression will be tested in transgenic mice. The results of these studies will provide basic information on the regulatory circuitry underlying fiber specificity, and will form the basis for building appropriate transgenic regulatory cassettes to effect fiber transitions in subsequent experimental manipulations on unweighted muscles.

Lrp5/6 are required for cerebellar development and for suppressing TH expression in Purkinje cells via β-catenin.

PubMed

Huang, Ying; Zhang, Qiong; Song, Ning-Ning; Zhang, Lei; Sun, Yu-Ling; Hu, Ling; Chen, Jia-Ying; Zhu, Weidong; Li, Jue; Ding, Yu-Qiang

2016-01-15

The cerebellum is responsible for coordinating motor functions and has a unique laminated architecture. Purkinje cells are inhibitory neurons and represent the only output from the cerebellar cortex. Tyrosine hydroxylase (TH) is the key enzyme for the synthesis of catecholamines, including dopamine and noradrenaline, and it is normally not expressed in cerebellar neurons. We report here that the low-density lipoprotein receptors (Lrp) 5 and 6, Wnt co-receptors, are required for the development of the cerebellum and for suppressing ectopic TH expression in Purkinje cells. Simultaneous inactivation of Lrp 5 and 6 by Nestin-Cre results in defective lamination and foliation of the cerebellum during postnatal development. Surprisingly, TH is ectopically expressed by Purkinje cells, although they still keep its other neurochemical characteristics. These phenotypes are also observed in the cerebellum of GFAP-Cre;β-catenin(flox/flox) mice, and AAV2-Cre-mediated gene deletion leads to ectopic TH expression in Purkinje cells of β-catenin(flox/flox) mice as well. Our results revealed a new role of the canonical Lrp5/6-β-catenin pathway in regulating the morphogenesis of the cerebellum during postnatal development.

Molecular and functional interactions between AKT and SOX2 in breast carcinoma

PubMed Central

Mir, Perihan; Konantz, Martina; Pereboom, Tamara C.; Paczulla, Anna M.; Merz, Britta; Fehm, Tanja; Perner, Sven; Rothfuss, Oliver C.; Kanz, Lothar; Schulze-Osthoff, Klaus; Lengerke, Claudia

2015-01-01

The transcription factor SOX2 is a key regulator of pluripotency in embryonic stem cells and plays important roles in early organogenesis. Recently, SOX2 expression was documented in various cancers and suggested as a cancer stem cell (CSC) marker. Here we identify the Ser/Thr-kinase AKT as an upstream regulator of SOX2 protein turnover in breast carcinoma (BC). SOX2 and pAKT are co-expressed and co-regulated in breast CSCs and depletion of either reduces clonogenicity. Ectopic SOX2 expression restores clonogenicity and in vivo tumorigenicity of AKT-inhibited cells, suggesting that SOX2 acts as a functional downstream AKT target. Mechanistically, we show that AKT physically interacts with the SOX2 protein to modulate its subcellular distribution. AKT kinase inhibition results in enhanced cytoplasmic retention of SOX2, presumably via impaired nuclear import, and in successive cytoplasmic proteasomal degradation of the protein. In line, blockade of either nuclear transport or proteasomal degradation rescues SOX2 expression in AKT-inhibited BC cells. Finally, AKT inhibitors efficiently suppress the growth of SOX2-expressing putative cancer stem cells, whereas conventional chemotherapeutics select for this population. Together, our results suggest the AKT/SOX2 molecular axis as a regulator of BC clonogenicity and AKT inhibitors as promising drugs for the treatment of SOX2-positive BC. PMID:26498353

Restrictions in Cell Cycle Progression of Adult Vestibular Supporting Cells in Response to Ectopic Cyclin D1 Expression

PubMed Central

Loponen, Heidi; Ylikoski, Jukka; Albrecht, Jeffrey H.; Pirvola, Ulla

2011-01-01

Sensory hair cells and supporting cells of the mammalian inner ear are quiescent cells, which do not regenerate. In contrast, non-mammalian supporting cells have the ability to re-enter the cell cycle and produce replacement hair cells. Earlier studies have demonstrated cyclin D1 expression in the developing mouse supporting cells and its downregulation along maturation. In explant cultures of the mouse utricle, we have here focused on the cell cycle control mechanisms and proliferative potential of adult supporting cells. These cells were forced into the cell cycle through adenoviral-mediated cyclin D1 overexpression. Ectopic cyclin D1 triggered robust cell cycle re-entry of supporting cells, accompanied by changes in p27Kip1 and p21Cip1 expressions. Main part of cell cycle reactivated supporting cells were DNA damaged and arrested at the G2/M boundary. Only small numbers of mitotic supporting cells and rare cells with signs of two successive replications were found. Ectopic cyclin D1-triggered cell cycle reactivation did not lead to hyperplasia of the sensory epithelium. In addition, a part of ectopic cyclin D1 was sequestered in the cytoplasm, reflecting its ineffective nuclear import. Combined, our data reveal intrinsic barriers that limit proliferative capacity of utricular supporting cells. PMID:22073316

Restrictions in cell cycle progression of adult vestibular supporting cells in response to ectopic cyclin D1 expression.

PubMed

Loponen, Heidi; Ylikoski, Jukka; Albrecht, Jeffrey H; Pirvola, Ulla

2011-01-01

Sensory hair cells and supporting cells of the mammalian inner ear are quiescent cells, which do not regenerate. In contrast, non-mammalian supporting cells have the ability to re-enter the cell cycle and produce replacement hair cells. Earlier studies have demonstrated cyclin D1 expression in the developing mouse supporting cells and its downregulation along maturation. In explant cultures of the mouse utricle, we have here focused on the cell cycle control mechanisms and proliferative potential of adult supporting cells. These cells were forced into the cell cycle through adenoviral-mediated cyclin D1 overexpression. Ectopic cyclin D1 triggered robust cell cycle re-entry of supporting cells, accompanied by changes in p27(Kip1) and p21(Cip1) expressions. Main part of cell cycle reactivated supporting cells were DNA damaged and arrested at the G2/M boundary. Only small numbers of mitotic supporting cells and rare cells with signs of two successive replications were found. Ectopic cyclin D1-triggered cell cycle reactivation did not lead to hyperplasia of the sensory epithelium. In addition, a part of ectopic cyclin D1 was sequestered in the cytoplasm, reflecting its ineffective nuclear import. Combined, our data reveal intrinsic barriers that limit proliferative capacity of utricular supporting cells.

Nuclear factor one B (NFIB) encodes a subtype-specific tumour suppressor in glioblastoma

PubMed Central

Stringer, Brett W.; Bunt, Jens; Day, Bryan W.; Barry, Guy; Jamieson, Paul R.; Ensbey, Kathleen S.; Bruce, Zara C.; Goasdoué, Kate; Vidal, Hélène; Charmsaz, Sara; Smith, Fiona M.; Cooper, Leanne T.; Piper, Michael

2016-01-01

Glioblastoma (GBM) is an essentially incurable and rapidly fatal cancer, with few markers predicting a favourable prognosis. Here we report that the transcription factor NFIB is associated with significantly improved survival in GBM. NFIB expression correlates inversely with astrocytoma grade and is lowest in mesenchymal GBM. Ectopic expression of NFIB in low-passage, patient-derived classical and mesenchymal subtype GBM cells inhibits tumourigenesis. Ectopic NFIB expression activated phospho-STAT3 signalling only in classical and mesenchymal GBM cells, suggesting a mechanism through which NFIB may exert its context-dependent tumour suppressor activity. Finally, NFIB expression can be induced in GBM cells by drug treatment with beneficial effects. PMID:27083054

BREAST CANCER-INDUCED BONE REMODELING, SKELETAL PAIN AND SPROUTING OF SENSORY NERVE FIBERS

PubMed Central

Bloom, Aaron P.; Jimenez-Andrade, Juan M.; Taylor, Reid N.; Castañeda-Corral, Gabriela; Kaczmarska, Magdalena J.; Freeman, Katie T.; Coughlin, Kathleen A.; Ghilardi, Joseph R.; Kuskowski, Michael A.; Mantyh, Patrick W.

2011-01-01

Breast cancer metastasis to bone is frequently accompanied by pain. What remains unclear is why this pain tends to become more severe and difficult to control with disease progression. Here we test the hypothesis that with disease progression sensory nerve fibers that innervate the breast cancer bearing bone undergo a pathological sprouting and reorganization, which in other non-malignant pathologies has been shown to generate and maintain chronic pain. Injection of human breast cancer cells (MDA-MB-231-BO) into the femoral intramedullary space of female athymic nude mice induces sprouting of calcitonin gene-related peptide (CGRP+) sensory nerve fibers. Nearly all CGRP+ nerve fibers that undergo sprouting also co-express tropomyosin receptor kinase A (TrkA+) and growth associated protein-43 (GAP43+). This ectopic sprouting occurs in periosteal sensory nerve fibers that are in close proximity to breast cancer cells, tumor-associated stromal cells and remodeled cortical bone. Therapeutic treatment with an antibody that sequesters nerve growth factor (NGF), administered when the pain and bone remodeling were first observed, blocks this ectopic sprouting and attenuates cancer pain. The present data suggest that the breast cancer cells and tumor-associated stromal cells express and release NGF, which drives bone pain and the pathological reorganization of nearby CGRP+ / TrkA+ / GAP43+ sensory nerve fibers. PMID:21497141

Aurora A regulates the activity of HURP by controlling the accessibility of its microtubule-binding domain.

PubMed

Wong, Jim; Lerrigo, Robert; Jang, Chang-Young; Fang, Guowei

2008-05-01

HURP is a spindle-associated protein that mediates Ran-GTP-dependent assembly of the bipolar spindle and promotes chromosome congression and interkinetochore tension during mitosis. We report here a biochemical mechanism of HURP regulation by Aurora A, a key mitotic kinase that controls the assembly and function of the spindle. We found that HURP binds to microtubules through its N-terminal domain that hyperstabilizes spindle microtubules. Ectopic expression of this domain generates defects in spindle morphology and function that reduce the level of tension across sister kinetochores and activate the spindle checkpoint. Interestingly, the microtubule binding activity of this N-terminal domain is regulated by the C-terminal region of HURP: in its hypophosphorylated state, C-terminal HURP associates with the microtubule-binding domain, abrogating its affinity for microtubules. However, when the C-terminal domain is phosphorylated by Aurora A, it no longer binds to N-terminal HURP, thereby releasing the inhibition on its microtubule binding and stabilizing activity. In fact, ectopic expression of this C-terminal domain depletes endogenous HURP from the mitotic spindle in HeLa cells in trans, suggesting the physiological importance for this mode of regulation. We concluded that phosphorylation of HURP by Aurora A provides a regulatory mechanism for the control of spindle assembly and function.

Evidence for the ectopic synthesis of melanin in human adipose tissue.

PubMed

Randhawa, Manpreet; Huff, Tom; Valencia, Julio C; Younossi, Zobair; Chandhoke, Vikas; Hearing, Vincent J; Baranova, Ancha

2009-03-01

Melanin is a common pigment in animals. In humans, melanin is produced in melanocytes, in retinal pigment epithelium (RPE) cells, in the inner ear, and in the central nervous system. Previously, we noted that human adipose tissue expresses several melanogenesis-related genes. In the current study, we confirmed the expression of melanogenesis-related mRNAs and proteins in human adipose tissue using real-time polymerase chain reaction and immunohistochemical staining. TYR mRNA signals were also detected by in situ hybridization in visceral adipocytes. The presence of melanin in human adipose tissue was revealed both by Fontana-Masson staining and by permanganate degradation of melanin coupled with liquid chromatography/ultraviolet/mass spectrometry determination of the pyrrole-2,3,5-tricarboxylic acid (PTCA) derivative of melanin. We also compared melanogenic activities in adipose tissues and in other human tissues using the L-[U-(14)C] tyrosine assay. A marked heterogeneity in the melanogenic activities of individual adipose tissue extracts was noted. We hypothesize that the ectopic synthesis of melanin in obese adipose may serve as a compensatory mechanism that uses its anti-inflammatory and its oxidative damage-absorbing properties. In conclusion, our study demonstrates for the first time that the melanin biosynthesis pathway is functional in adipose tissue.

Osteophyte formation and matrix mineralization in a TMJ osteoarthritis mouse model are associated with ectopic hedgehog signaling

PubMed Central

Bechtold, Till E.; Saunders, Cheri; Decker, Rebekah S.; Um, Hyo-Bin; Cottingham, Naiga; Salhab, Imad; Kurio, Naito; Billings, Paul C.; Pacifici, Maurizio; Nah, Hyun-Duck; Koyama, Eiki

2016-01-01

The temporomandibular joint (TMJ) is a diarthrodial joint that relies on lubricants for frictionless movement and long-term function. It remains unclear what temporal and causal relationships may exist between compromised lubrication and onset and progression of TMJ disease. Here we report that Proteoglycan 4 (Prg4)-null TMJs exhibit irreversible osteoarthritis-like changes over time and are linked to formation of ectopic mineralized tissues and osteophytes in articular disc, mandibular condyle and glenoid fossa. In the presumptive layer of mutant glenoid fossa’s articulating surface, numerous chondrogenic cells and/or chondrocytes emerged ectopically within the type I collagen-expressing cell population, underwent endochondral bone formation accompanied by enhanced Ihh expression, became entrapped into temporal bone mineralized matrix, and thereby elicited excessive chondroid bone formation. As the osteophytes grew, the roof of the glenoid fossa/eminence became significantly thicker and flatter, resulting in loss of its characteristic concave shape for accommodation of condyle and disc. Concurrently, the condyles became flatter and larger and exhibited ectopic bone along their neck, likely supporting the enlarged condylar heads. Articular discs lost their concave configuration, and ectopic cartilage developed and articulated with osteophytes. In glenoid fossa cells in culture, hedgehog signaling stimulated chondrocyte maturation and mineralization including alkaline phosphatase, while treatment with hedgehog inhibitor HhAntag prevented such maturation process. In sum, our data indicate that Prg4 is needed for TMJ integrity and long-term postnatal function. In its absence, progenitor cells near presumptive articular layer and disc undergo ectopic chondrogenesis and generate ectopic cartilage, possibly driven by aberrant activation of Hh signaling. The data suggest also that the Prg4-null mice represent a useful model to study TMJ osteoarthritis-like degeneration and clarify its pathogenesis. PMID:26945615

Ectopic expression of homeobox gene NKX2-1 in diffuse large B-cell lymphoma is mediated by aberrant chromatin modifications.

PubMed

Nagel, Stefan; Ehrentraut, Stefan; Tomasch, Jürgen; Quentmeier, Hilmar; Meyer, Corinna; Kaufmann, Maren; Drexler, Hans G; MacLeod, Roderick A F

2013-01-01

Homeobox genes encode transcription factors ubiquitously involved in basic developmental processes, deregulation of which promotes cell transformation in multiple cancers including hematopoietic malignancies. In particular, NKL-family homeobox genes TLX1, TLX3 and NKX2-5 are ectopically activated by chromosomal rearrangements in T-cell neoplasias. Here, using transcriptional microarray profiling and RQ-PCR we identified ectopic expression of NKL-family member NKX2-1, in a diffuse large B-cell lymphoma (DLBCL) cell line SU-DHL-5. Moreover, in silico analysis demonstrated NKX2-1 overexpression in 5% of examined DLBCL patient samples. NKX2-1 is physiologically expressed in lung and thyroid tissues where it regulates differentiation. Chromosomal and genomic analyses excluded rearrangements at the NKX2-1 locus in SU-DHL-5, implying alternative activation. Comparative expression profiling implicated several candidate genes in NKX2-1 regulation, variously encoding transcription factors, chromatin modifiers and signaling components. Accordingly, siRNA-mediated knockdown and overexpression studies confirmed involvement of transcription factor HEY1, histone methyltransferase MLL and ubiquitinated histone H2B in NKX2-1 deregulation. Chromosomal aberrations targeting MLL at 11q23 and the histone gene cluster HIST1 at 6p22 which we observed in SU-DHL-5 may, therefore, represent fundamental mutations mediating an aberrant chromatin structure at NKX2-1. Taken together, we identified ectopic expression of NKX2-1 in DLBCL cells, representing the central player in an oncogenic regulative network compromising B-cell differentiation. Thus, our data extend the paradigm of NKL homeobox gene deregulation in lymphoid malignancies.

Inhibition of mitogen-activated protein kinase Erk1/2 promotes protein degradation of ATP binding cassette transporters A1 and G1 in CHO and HuH7 cells.

PubMed

Mulay, Vishwaroop; Wood, Peta; Manetsch, Melanie; Darabi, Masoud; Cairns, Rose; Hoque, Monira; Chan, Karen Cecilia; Reverter, Meritxell; Alvarez-Guaita, Anna; Rye, Kerry-Anne; Rentero, Carles; Heeren, Joerg; Enrich, Carlos; Grewal, Thomas

2013-01-01

Signal transduction modulates expression and activity of cholesterol transporters. We recently demonstrated that the Ras/mitogen-activated protein kinase (MAPK) signaling cascade regulates protein stability of Scavenger Receptor BI (SR-BI) through Proliferator Activator Receptor (PPARα) -dependent degradation pathways. In addition, MAPK (Mek/Erk 1/2) inhibition has been shown to influence liver X receptor (LXR) -inducible ATP Binding Cassette (ABC) transporter ABCA1 expression in macrophages. Here we investigated if Ras/MAPK signaling could alter expression and activity of ABCA1 and ABCG1 in steroidogenic and hepatic cell lines. We demonstrate that in Chinese Hamster Ovary (CHO) cells and human hepatic HuH7 cells, extracellular signal-regulated kinase 1/2 (Erk1/2) inhibition reduces PPARα-inducible ABCA1 protein levels, while ectopic expression of constitutively active H-Ras, K-Ras and MAPK/Erk kinase 1 (Mek1) increases ABCA1 protein expression, respectively. Furthermore, Mek1/2 inhibitors reduce ABCG1 protein levels in ABCG1 overexpressing CHO cells (CHO-ABCG1) and human embryonic kidney 293 (HEK293) cells treated with LXR agonist. This correlates with Mek1/2 inhibition reducing ABCG1 cell surface expression and decreasing cholesterol efflux onto High Density Lipoproteins (HDL). Real Time reverse transcriptase polymerase chain reaction (RT-PCR) and protein turnover studies reveal that Mek1/2 inhibitors do not target transcriptional regulation of ABCA1 and ABCG1, but promote ABCA1 and ABCG1 protein degradation in HuH7 and CHO cells, respectively. In line with published data from mouse macrophages, blocking Mek1/2 activity upregulates ABCA1 and ABCG1 protein levels in human THP1 macrophages, indicating opposite roles for the Ras/MAPK pathway in the regulation of ABC transporter activity in macrophages compared to steroidogenic and hepatic cell types. In summary, this study suggests that Ras/MAPK signaling modulates PPARα- and LXR-dependent protein degradation pathways in a cell-specific manner to regulate the expression levels of ABCA1 and ABCG1 transporters.

The HMGA proteins: a myriad of functions (Review).

PubMed

Cleynen, Isabelle; Van de Ven, Wim J M

2008-02-01

The 'high mobility group' HMGA protein family consists of four members: HMGA1a, HMGA1b and HMGA1c, which result from translation of alternative spliced forms of one gene and HMGA2, which is encoded for by another gene. HMGA proteins are characterized by three DNA-binding domains, called AT-hooks, and an acidic carboxy-terminal tail. HMGA proteins are architectural transcription factors that both positively and negatively regulate the transcription of a variety of genes. They do not display direct transcriptional activation capacity, but regulate gene expression by changing the DNA conformation by binding to AT-rich regions in the DNA and/or direct interaction with several transcription factors. In this way, they influence a diverse array of normal biological processes including cell growth, proliferation, differentiation and death. Both HMGA1 and HMGA2 are hardly detectable in normal adult tissue but are abundantly and ubiquitously expressed during embryonic development. In malignant epithelial tumors as well as in leukemia, however, expression of HMGA1 is again strongly elevated to embryonic levels thus leading to ectopic expression of (fetal) target genes. HMGA2 overexpression also has a causal role in inducing neoplasia. Besides overexpression of full length HMGA proteins in different tumors, the HMGA genes are often involved in chromosomal rearrangements. Such translocations are mostly detected in benign tumors of mesenchymal origin and are believed to be one of the most common chromosomal rearrangements in human neoplasia. To provide clarity in the abundance of articles on this topic, this review gives a general overview of the nuclear functions and regulation of the HMGA genes and corresponding proteins.

The zinc finger gene Krox20 regulates HoxB2 (Hox2.8) during hindbrain segmentation.

PubMed

Sham, M H; Vesque, C; Nonchev, S; Marshall, H; Frain, M; Gupta, R D; Whiting, J; Wilkinson, D; Charnay, P; Krumlauf, R

1993-01-29

The zinc finger gene Krox20 and many Hox homeobox genes are expressed in segment-restricted domains in the hindbrain. The restricted expression patterns appear before morphological segmentation, suggesting that these transcription factors may play an early role in the establishment and identity of rhombomeric segments. In this paper, we show that the HoxB2 (Hox2.8) gene is normally upregulated in rhombomeres (r) 3, 4, and 5, and we identify an enhancer region upstream of the gene that imposes r3/r5 expression in transgenic mice. This enhancer contains three Krox20-binding sites required in vitro for complex formation with Krox20 protein and in vivo for rhombomere-restricted expression. In transgenic mice, Krox20 expressed in ectopic domains can transactivate a reporter construct containing the HoxB2 r3/r5 enhancer. These data demonstrate that Krox20 is a part of the upstream transcriptional cascade that directly regulates HoxB2 expression during hindbrain segmentation.

Neurologic impairment from ectopic bone in the lumbar canal: a potential complication of off-label PLIF/TLIF use of bone morphogenetic protein-2 (BMP-2).

PubMed

Wong, David A; Kumar, Anant; Jatana, Sanjay; Ghiselli, Gary; Wong, Katherine

2008-01-01

Bone morphogenetic protein-2 (BMP-2) (Infuse) has been approved for use in anterior lumbar fusion in conjunction with an LT cage. However, off-label use is seen with anterior cervical fusion, posterior lumbar interbody fusion (PLIF), and transforaminal lumbar interbody fusion (TLIF). The Federal Food and Drug Administration trial of BMP-2 in a PLIF application was halted because of a high incidence of ectopic bone forming in the neural canal (75%). The authors did not find a correlation between ectopic bone and increased leg pain. They concluded that the ectopic bone was a radiographic phenomenon and not associated with clinical findings. Complications using BMP in the cervical spine have been reported. Heretofore, there has not been a similar warning voiced for use of BMP in a lumbar PLIF or TLIF. The purpose was to report five cases of ectopic bone in the canal associated with PLIF/TLIF off-label use of BMP-2 potentially contributing to abnormal neurologic findings. This is an observational cohort study of patients referred to a tertiary care private medical center. This was a retrospective chart review of patients referred to a tertiary spine institute with complications after surgery where BMP-2 had been used in an off-label PLIF or TLIF application. Patient demographics, operating room (OR) notes from the index BMP surgery, imaging studies, and current clinical status were reviewed. Five cases of ectopic bone in the spinal canal with potential neurologic compromise were identified. It does appear that ectopic bone in the spinal canal associated with BMP-2 use in PLIF or TLIF may contribute to symptomatic neurologic findings in rare cases. Revision surgeries are difficult. This article challenges a previous publication, which concluded that the high incidence of ectopic bone was of no clinical significance. Isolating BMP anteriorly in the disc space using layered barriers of bone graft between the BMP and the annular defect may reduce the incidence of ectopic bone in the spinal canal. Surgeons need to weigh the benefits versus risks of any technology used off label when making treatment decisions with their patients.

Growth enhancement in transgenic tilapia by ectopic expression of tilapia growth hormone.

PubMed

Martínez, R; Estrada, M P; Berlanga, J; Guillén, I; Hernández, O; Cabrera, E; Pimentel, R; Morales, R; Herrera, F; Morales, A; Piña, J C; Abad, Z; Sánchez, V; Melamed, P; Lleonart, R; de la Fuente, J

1996-03-01

The generation of transgenic fish with the transfer of growth hormone (GH) genes has opened new possibilities for the manipulation of growth in economically important fish species. The tilapia growth hormone (tiGH) cDNA was linked to the human cytomegalovirus (CMV) enhancer-promoter and used to generate transgenic tilapia by microinjection into one-cell embryos. Five transgenic tilapia were obtained from 40 injected embryos. A transgenic animal containing one copy of the transgene per cell was selected to establish a transgenic line. The transgene was stably transmitted to F1 and F2 generations in a Mendelian fashion. Ectopic, low-level expression of tiGH was detected in gonad and muscle cells of F1 transgenic tilapia by immunohystochemical analysis of tissue sections. Nine-month-old transgenic F1 progeny were 82% larger than nontransgenic fish at p = .001. These results showed that low-level ectopic expression of tiGH resulted in a growth acceleration in transgenic tilapia. Tilapia GH gene transfer is an alternative for growth acceleration in tilapia.

Ectopic Expression of Xylella fastidiosa rpfF Conferring Production of Diffusible Signal Factor in Transgenic Tobacco and Citrus Alters Pathogen Behavior and Reduces Disease Severity.

PubMed

Caserta, R; Souza-Neto, R R; Takita, M A; Lindow, S E; De Souza, A A

2017-11-01

The pathogenicity of Xylella fastidiosa is associated with its ability to colonize the xylem of host plants. Expression of genes contributing to xylem colonization are suppressed, while those necessary for insect vector acquisition are increased with increasing concentrations of diffusible signal factor (DSF), whose production is dependent on RpfF. We previously demonstrated that transgenic citrus plants ectopically expressing rpfF from a citrus strain of X. fastidiosa subsp. pauca exhibited less susceptibility to Xanthomonas citri subsp. citri, another pathogen whose virulence is modulated by DSF accumulation. Here, we demonstrate that ectopic expression of rpfF in both transgenic tobacco and sweet orange also confers a reduction in disease severity incited by X. fastidiosa and reduces its colonization of those plants. Decreased disease severity in the transgenic plants was generally associated with increased expression of genes conferring adhesiveness to the pathogen and decreased expression of genes necessary for active motility, accounting for the reduced population sizes achieved in the plants, apparently by limiting pathogen dispersal through the plant. Plant-derived DSF signal molecules in a host plant can, therefore, be exploited to interfere with more than one pathogen whose virulence is controlled by DSF signaling.

Pyruvate kinase type M2 promotes tumour cell exosome release via phosphorylating synaptosome-associated protein 23

PubMed Central

Wei, Yao; Wang, Dong; Jin, Fangfang; Bian, Zhen; Li, Limin; Liang, Hongwei; Li, Mingzhen; Shi, Lei; Pan, Chaoyun; Zhu, Dihan; Chen, Xi; Hu, Gang; Liu, Yuan; Zhang, Chen-Yu; Zen, Ke

2017-01-01

Tumour cells secrete exosomes that are involved in the remodelling of the tumour–stromal environment and promoting malignancy. The mechanisms governing tumour exosome release, however, remain incompletely understood. Here we show that tumour cell exosomes secretion is controlled by pyruvate kinase type M2 (PKM2), which is upregulated and phosphorylated in tumours. During exosome secretion, phosphorylated PKM2 serves as a protein kinase to phosphorylate synaptosome-associated protein 23 (SNAP-23), which in turn enables the formation of the SNARE complex to allow exosomes release. Direct phosphorylation assay and mass spectrometry confirm that PKM2 phosphorylates SNAP-23 at Ser95. Ectopic expression of non-phosphorylated SNAP-23 mutant (Ser95→Ala95) significantly reduces PKM2-mediated exosomes release whereas expression of selective phosphomimetic SNAP-23 mutants (Ser95→Glu95 but not Ser20→Glu20) rescues the impaired exosomes release induced by PKM2 knockdown. Our findings reveal a non-metabolic function of PKM2, an enzyme associated with tumour cell reliance on aerobic glycolysis, in promoting tumour cell exosome release. PMID:28067230

Cutting Edge: Inflammasome Activation in Primary Human Macrophages Is Dependent on Flagellin

PubMed Central

Kortmann, Jens; Brubaker, Sky W.

2015-01-01

Murine NLR family, apoptosis inhibitory protein (Naip)1, Naip2, and Naip5/6 are host sensors that detect the cytosolic presence of needle and rod proteins from bacterial type III secretion systems and flagellin, respectively. Previous studies using human-derived macrophage-like cell lines indicate that human macrophages sense the cytosolic needle protein, but not bacterial flagellin. In this study, we show that primary human macrophages readily sense cytosolic flagellin. Infection of primary human macrophages with Salmonella elicits robust cell death and IL-1β secretion that is dependent on flagellin. We show that flagellin detection requires a full-length isoform of human Naip. This full-length Naip isoform is robustly expressed in primary macrophages from healthy human donors, but it is drastically reduced in monocytic tumor cells, THP-1, and U937, rendering them insensitive to cytosolic flagellin. However, ectopic expression of full-length Naip rescues the ability of U937 cells to sense flagellin. In conclusion, human Naip functions to activate the inflammasome in response to flagellin, similar to murine Naip5/6. PMID:26109648

The Homothorax homeoprotein activates the nuclear localization of another homeoprotein, Extradenticle, and suppresses eye development in Drosophila

PubMed Central

Pai, Chi-Yun; Kuo, Tung-Sheng; Jaw, Thomas J.; Kurant, Estee; Chen, Cheng-Tse; Bessarab, Dmitri A.; Salzberg, Adi; Sun, Y. Henry

1998-01-01

The Extradenticle (Exd) protein in Drosophila acts as a cofactor to homeotic proteins. Its nuclear localization is regulated. We report the cloning of the Drosophila homothorax (hth) gene, a homolog of the mouse Meis1 proto-oncogene that has a homeobox related to that of exd. Comparison with Meis1 finds two regions of high homology: a novel MH domain and the homeodomain. In imaginal discs, hth expression coincides with nuclear Exd. hth and exd also have virtually identical, mutant clonal phenotypes in adults. These results suggest that hth and exd function in the same pathway. We show that hth acts upstream of exd and is required and sufficient for Exd protein nuclear localization. We also show that hth and exd are both negative regulators of eye development; their mutant clones caused ectopic eye formation. Targeted expression of hth, but not of exd, in the eye disc abolished eye development completely. We suggest that hth acts with exd to delimit the eye field and prevent inappropriate eye development. PMID:9450936

Cytoskeleton-interacting LIM-domain protein CRP1 suppresses cell proliferation and protects from stress-induced cell death

DOE Office of Scientific and Technical Information (OSTI.GOV)

Latonen, Leena; Jaervinen, Paeivi M.; Haartman Institute, University of Helsinki, FIN-00014 Helsinki

2008-02-15

Members of the cysteine-rich protein (CRP) family are actin cytoskeleton-interacting LIM-domain proteins known to act in muscle cell differentiation. We have earlier found that CRP1, a founding member of this family, is transcriptionally induced by UV radiation in human diploid fibroblasts [M. Gentile, L. Latonen, M. Laiho, Cell cycle arrest and apoptosis provoked by UV radiation-induced DNA damage are transcriptionally highly divergent responses, Nucleic Acids Res. 31 (2003) 4779-4790]. Here we show that CRP1 is induced by growth-inhibitory signals, such as increased cellular density, and cytotoxic stress induced by UV radiation or staurosporine. We found that high levels of CRP1more » correlate with differentiation-associated morphology towards the myofibroblast lineage and that expression of ectopic CRP1 suppresses cell proliferation. Following UV- and staurosporine-induced stresses, expression of CRP1 provides a survival advantage evidenced by decreased cellular death and increased cellular metabolic activity and attachment. Our studies identify that CRP1 is a novel stress response factor, and provide evidence for its growth-inhibitory and cytoprotective functions.« less

Co-expression of the transcription factors CEH-14 and TTX-1 regulates AFD neuron-specific genes gcy-8 and gcy-18 in C. elegans.

PubMed

Kagoshima, Hiroshi; Kohara, Yuji

2015-03-15

A wide variety of cells are generated by the expression of characteristic sets of genes, primarily those regulated by cell-specific transcription. To elucidate the mechanism regulating cell-specific gene expression in a highly specialized cell, AFD thermosensory neuron in Caenorhabditis elegans, we analyzed the promoter sequences of guanylyl cyclase genes, gcy-8 and gcy-18, exclusively expressed in AFD. In this study, we showed that AFD-specific expression of gcy-8 and gcy-18 requires the co-expression of homeodomain proteins, CEH-14/LHX3 and TTX-1/OTX1. We observed that mutation of ttx-1 or ceh-14 caused a reduction in the expression of gcy-8 and gcy-18 and that the expression was completely lost in double mutants. This synergy effect was also observed with other AFD marker genes, such as ntc-1, nlp-21and cng-3. Electrophoretic mobility shift assays revealed direct interaction of CEH-14 and TTX-1 proteins with gcy-8 and gcy-18 promoters in vitro. The binding sites of CEH-14 and TTX-1 proteins were confirmed to be essential for AFD-specific expression of gcy-8 and gcy-18 in vivo. We also demonstrated that forced expression of CEH-14 and TTX-1 in AWB chemosensory neurons induced ectopic expression of gcy-8 and gcy-18 reporters in this neuron. Finally, we showed that the regulation of gcy-8 and gcy-18 expression by ceh-14 and ttx-1 is evolutionally conserved in five Caenorhabditis species. Taken together, ceh-14 and ttx-1 expression determines the fate of AFD as terminal selector genes at the final step of cell specification. Copyright © 2015 Elsevier Inc. All rights reserved.

Phospholipase C/protein kinase C pathway mediates angiotensin II-dependent apoptosis in neonatal rat cardiac fibroblasts expressing AT1 receptor.

PubMed

Vivar, Raul; Soto, Cristian; Copaja, Miguel; Mateluna, Francisca; Aranguiz, Pablo; Muñoz, Juan Pablo; Chiong, Mario; Garcia, Lorena; Letelier, Alan; Thomas, Walter G; Lavandero, Sergio; Díaz-Araya, Guillermo

2008-08-01

Cardiac fibroblasts are the major non-myocyte cell constituent in the myocardium, and they are involved in heart remodeling. Angiotensin II type 1 receptor (AT1R) mediates the established actions of angiotensin II (Ang II), and changes in its expression have been reported in cardiac fibroblasts after myocardial infarction. However, the AT1R-dependent signaling pathways involved in cardiac fibroblast death remain unknown. Using adenovirus, we ectopically expressed AT1R in cultured neonatal rat cardiac fibroblasts and investigated the role of the phospholipase (PLC)/protein kinase C (PKC) pathway on Ang II-dependent death. Ang II induced cardiac fibroblast death characterized by an early loss of mitochondrial membrane potential, increased Bax/Bcl-2 ratio, caspase-3 activation, and DNA fragmentation. All these effects were prevented by the AT1R antagonist losartan, PLC inhibitor U73122, and PKC inhibitor Gö6976. We conclude that Ang II stimulates the intrinsic apoptotic pathway in cultured cardiac fibroblasts by the AT1R/PLC/PKC signaling pathway.

P-body-induced inactivation of let-7a miRNP prevents the death of growth factor-deprived neuronal cells.

PubMed

Patranabis, Somi; Bhattacharyya, Suvendra Nath

2018-03-01

RNA processing bodies (P-bodies) are cytoplasmic RNA granules in eukaryotic cells that regulate gene expression by executing the translation suppression and degradation of mRNAs that are targeted to these bodies. P-bodies can also serve as storage sites for translationally repressed mRNAs both in mammalian cells and yeast cells. In this report, a unique role of mammalian P-bodies is documented. Depletion of P-body components dedifferentiate nerve growth factor-treated PC12 cells, whereas ectopic expression of P-body components induces the neuronal differentiation of precursor cells. Trophic factor withdrawal from differentiated cells induces a decrease in cellular P-body size and numbers that are coupled with dedifferentiation and cell death. Here, we report how the expression of P-body proteins-by ensuring the phosphorylation of argonaute protein 2 and the subsequent inactivation let-7a miRNPs-prevents the apoptotic death of growth factor-depleted neuronal cells.-Patranabis, S., Bhattacharyya, S. N. P-body-induced inactivation of let-7a miRNP prevents the death of growth factor-deprived neuronal cells.

Drosophila Myc is oncogenic in mammalian cells and plays a role in the diminutive phenotype

PubMed Central

Schreiber-Agus, Nicole; Stein, David; Chen, Ken; Goltz, Jason S.; Stevens, Leslie; DePinho, Ronald A.

1997-01-01

Biochemical and biological activities of Myc oncoproteins are highly dependent upon their association with another basic region helix–loop–helix/leucine zipper (bHLH/LZ) protein, Max. Our previous observation that the DNA-binding/dimerization region of Max is absolutely conserved throughout vertebrate evolution provided the basis for a yeast two-hybrid interaction screen that led to the isolation of the Drosophila Myc (dMyc1) protein. Structural conservation in regions of known functional significance is consistent with the ability of dMyc1 to interact with vertebrate Max, to transactivate gene expression in yeast cells, and to cooperate with activated H-RAS to effect the malignant transformation of primary mammalian cells. The ability of P-element-mediated ectopic expression of dmyc1 to reverse a subset of the phenotypic alterations associated with the diminutive mutation suggests that diminutive may correspond to dmyc1. This finding, along with the localization of dmyc1 expression to zones of high proliferative activity in the embryo, implicates dMyc1 as an integral regulator of Drosophila growth and development. PMID:9037036

The R2R3MYB VvMYBPA1 from grape reprograms the phenylpropanoid pathway in tobacco flowers.

PubMed

Passeri, Valentina; Martens, Stefan; Carvalho, Elisabete; Bianchet, Chantal; Damiani, Francesco; Paolocci, Francesco

2017-08-01

This work shows that, in tobacco, the ectopic expression of VvMYBPA1 , a grape regulator of proanthocyanidin biosynthesis, up- or down-regulates different branches of the phenylproanoid pathway, in a structure-specific fashion. Proanthocyanidins are flavonoids of paramount importance for animal and human diet. Research interest increasingly tilts towards generating crops enriched with these health-promoting compounds. Flavonoids synthesis is regulated by the MBW transcriptional complex, made of R2R3MYB, bHLH and WD40 proteins, with the MYB components liable for channeling the complex towards specific branches of the pathway. Hence, using tobacco as a model, here, we tested if the ectopic expression of the proanthocyanidin regulator VvMYBPA1 from grape induces the biosynthesis of these compounds in not-naturally committed cells. Here, we show, via targeted transcriptomic and metabolic analyses of primary transgenic lines and their progeny, that VvMYBPA1 alters the phenylpropanoid pathway in tobacco floral organs, in a structure-specific fashion. We also report that a modest VvMYBPA1 expression is sufficient to induce the expression of both proanthocyanidin-specific and early genes of the phenylpropanoid pathway. Consequently, proanthocyanidins and chlorogenic acids are induced or de novo synthetised in floral limbs, tubes and stamens. Other phenylpropanoid branches are conversely induced or depleted according to the floral structure. Our study documents a novel and distinct function of VvMYBPA1 with respect to other MYBs regulating proanthocyanidins. Present findings may have major implications in designing strategies for enriching crops with health-promoting compounds.

Aldo-keto reductase family 1 B10 protein detoxifies dietary and lipid-derived alpha, beta-unsaturated carbonyls at physiological levels.

PubMed

Zhong, Linlin; Liu, Ziwen; Yan, Ruilan; Johnson, Stephen; Zhao, Yupei; Fang, Xiubin; Cao, Deliang

2009-09-18

Alpha, beta-unsaturated carbonyls are highly reactive mutagens and carcinogens to which humans are exposed on a daily basis. This study demonstrates that aldo-keto reductase family 1 member B10 (AKR1B10) is a critical protein in detoxifying dietary and lipid-derived unsaturated carbonyls. Purified AKR1B10 recombinant protein efficiently catalyzed the reduction to less toxic alcohol forms of crotonaldehyde at 0.90 microM, 4-hydroxynonenal (HNE) at 0.10 microM, trans-2-hexanal at 0.10 microM, and trans-2,4-hexadienal at 0.05 microM, the concentrations at or lower than physiological exposures. Ectopically expressed AKR1B10 in 293T cells eliminated immediately HNE at 1 (subtoxic) or 5 microM (toxic) by converting to 1,4-dihydroxynonene, protecting the cells from HNE toxicity. AKR1B10 protein also showed strong enzymatic activity toward glutathione-conjugated carbonyls. Taken together, our study results suggest that AKR1B10 specifically expressed in the intestine is physiologically important in protecting the host cell against dietary and lipid-derived cytotoxic carbonyls.

Aldo-keto reductase family 1 B10 protein detoxifies dietary and lipid-derived alpha, beta-unsaturated carbonyls at physiological levels

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhong, Linlin; Department of Neurobiology and Anatomy, China Medical University, Shenyang 110001; Liu, Ziwen

2009-09-18

Alpha, beta-unsaturated carbonyls are highly reactive mutagens and carcinogens to which humans are exposed on a daily basis. This study demonstrates that aldo-keto reductase family 1 member B10 (AKR1B10) is a critical protein in detoxifying dietary and lipid-derived unsaturated carbonyls. Purified AKR1B10 recombinant protein efficiently catalyzed the reduction to less toxic alcohol forms of crotonaldehyde at 0.90 {mu}M, 4-hydroxynonenal (HNE) at 0.10 {mu}M, trans-2-hexanal at 0.10 {mu}M, and trans-2,4-hexadienal at 0.05 {mu}M, the concentrations at or lower than physiological exposures. Ectopically expressed AKR1B10 in 293T cells eliminated immediately HNE at 1 (subtoxic) or 5 {mu}M (toxic) by converting to 1,4-dihydroxynonene,more » protecting the cells from HNE toxicity. AKR1B10 protein also showed strong enzymatic activity toward glutathione-conjugated carbonyls. Taken together, our study results suggest that AKR1B10 specifically expressed in the intestine is physiologically important in protecting the host cell against dietary and lipid-derived cytotoxic carbonyls.« less

Protection of the Photosynthetic Apparatus from Extreme Dehydration and Oxidative Stress in Seedlings of Transgenic Tobacco

PubMed Central

Personat, José-María; Tejedor-Cano, Javier; Lindahl, Marika; Diaz-Espejo, Antonio; Jordano, Juan

2012-01-01

A genetic program that in sunflower seeds is activated by Heat Shock transcription Factor A9 (HaHSFA9) has been analyzed in transgenic tobacco seedlings. The ectopic overexpression of the HSFA9 program protected photosynthetic membranes, which resisted extreme dehydration and oxidative stress conditions. In contrast, heat acclimation of seedlings induced thermotolerance but not resistance to the harsh stress conditions employed. The HSFA9 program was found to include the expression of plastidial small Heat Shock Proteins that accumulate only at lower abundance in heat-stressed vegetative organs. Photosystem II (PSII) maximum quantum yield was higher for transgenic seedlings than for non-transgenic seedlings, after either stress treatment. Furthermore, protection of both PSII and Photosystem I (PSI) membrane protein complexes was observed in the transgenic seedlings, leading to their survival after the stress treatments. It was also shown that the plastidial D1 protein, a labile component of the PSII reaction center, and the PSI core protein PsaB were shielded from oxidative damage and degradation. We infer that natural expression of the HSFA9 program during embryogenesis may protect seed pro-plastids from developmental desiccation. PMID:23227265

GLI1-mediated regulation of side population is responsible for drug resistance in gastric cancer

PubMed Central

Yu, Beiqin; Gu, Dongsheng; Zhang, Xiaoli; Li, Jianfang; Liu, Bingya; Xie, Jingwu

2017-01-01

Gastric cancer is the third leading cause of cancer-related mortality worldwide. Chemotherapy is frequently used for gastric cancer treatment. Most patients with advanced gastric cancer eventually succumb to the disease despite some patients responded initially to chemotherapy. Thus, identifying molecular mechanisms responsible for cancer relapse following chemotherapy will help design new ways to treat gastric cancer. In this study, we revealed that the residual cancer cells following treatment with chemotherapeutic reagent cisplatin have elevated expression of hedgehog target genes GLI1, GLI2 and PTCH1, suggestive of hedgehog signaling activation. We showed that GLI1 knockdown sensitized gastric cancer cells to CDDP whereas ectopic GLI1 expression decreased the sensitivity. Further analyses indicate elevated GLI1 expression is associated with an increase in tumor sphere formation, side population and cell surface markers for putative cancer stem cells. We have evidence to support that GLI1 is critical for maintenance of putative cancer stem cells through direct regulation of ABCG2. In fact, GLI1 protein was shown to be associated with the promoter fragment of ABCG2 through a Gli-binding consensus site in gastric cancer cells. Disruption of ABCG2 function, through ectopic expression of an ABCG2 dominant negative construct or a specific ABCG2 inhibitor, increased drug sensitivity of cancer cells both in culture and in mice. The relevance of our studies to gastric cancer patient care is reflected by our discovery that high ABCG2 expression was associated with poor survival in the gastric cancer patients who underwent chemotherapy. Taken together, we have identified a molecular mechanism by which gastric cancer cells gain chemotherapy resistance. PMID:28404967

F-Box Protein FBX92 Affects Leaf Size in Arabidopsis thaliana.

PubMed

Baute, Joke; Polyn, Stefanie; De Block, Jolien; Blomme, Jonas; Van Lijsebettens, Mieke; Inzé, Dirk

2017-05-01

F-box proteins are part of one of the largest families of regulatory proteins that play important roles in protein degradation. In plants, F-box proteins are functionally very diverse, and only a small subset has been characterized in detail. Here, we identified a novel F-box protein FBX92 as a repressor of leaf growth in Arabidopsis. Overexpression of AtFBX92 resulted in plants with smaller leaves than the wild type, whereas plants with reduced levels of AtFBX92 showed, in contrast, increased leaf growth by stimulating cell proliferation. Detailed cellular analysis suggested that AtFBX92 specifically affects the rate of cell division during early leaf development. This is supported by the increased expression levels of several cell cycle genes in plants with reduced AtFBX92 levels. Surprisingly, overexpression of the maize homologous gene ZmFBX92 in maize had no effect on plant growth, whereas ectopic expression in Arabidopsis increased leaf growth. Expression of a truncated form of AtFBX92 showed that the contrasting effects of ZmFBX92 and AtFBX92 gain of function in Arabidopsis are due to the absence of the F-box-associated domain in the ZmFBX92 gene. Our work reveals an additional player in the complex network that determines leaf size and lays the foundation for identifying putative substrates. © The Author 2017. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.

Unremitting Cell Proliferation in the Secretory Phase of Eutopic Endometriosis

PubMed Central

Franco-Murillo, Yanira; Miranda-Rodríguez, José Antonio; Rendón-Huerta, Erika; Montaño, Luis F.; Cornejo, Gerardo Velázquez; Gómez, Lucila Poblano; Valdez-Morales, Francisco Javier; Gonzalez-Sanchez, Ignacio

2014-01-01

Objective: Endometriosis is linked to altered cell proliferation and stem cell markers c-kit/stem cell factor (SCF) in ectopic endometrium. Our aim was to investigate whether c-kit/SCF also plays a role in eutopic endometrium. Design: Eutopic endometrium obtained from 35 women with endometriosis and 25 fertile eumenorrheic women was analyzed for in situ expression of SCF/c-kit, Ki67, RAC-alpha serine/threonine-protein kinase (Akt), phosphorylated RAC-alpha serine/threonin-protein kinase (pAkt), Glycogen synthase kinase 3 beta (GSK3β), and phosphorylated glycogen synthase kinase 3 beta (pGSK3β), throughout the menstrual cycle. Results: Expression of Ki67 and SCF was higher in endometriosis than in control tissue (P < .05) and greater in secretory rather than proliferative (P < .01) endometrium in endometriosis. Expression of c-kit was also higher in endometriosis although similar in both phases. Expression of Akt and GSK3β was identical in all samples and cycle phases, whereas pAkt and pGSK3β, opposed to control tissue, remained overexpressed in the secretory phase in endometriosis. Conclusion: Unceasing cell proliferation in the secretory phase of eutopic endometriosis is linked to deregulation of c-kit/SCF-associated signaling pathways. PMID:25194152

Absence of catalytic domain in a putative protein kinase C (PkcA) suppresses tip dominance in Dictyostelium discoideum

PubMed Central

Mohamed, Wasima; Ray, Sibnath; Brazill, Derrick; Baskar, Ramamurthy

2017-01-01

A number of organisms possess several isoforms of protein kinase C but little is known about the significance of any specific isoform during embryogenesis and development. To address this we characterized a PKC ortholog (PkcA; DDB_G0288147) in Dictyostelium discoideum. pkcA expression switches from prestalk in mound to prespore in slug, indicating a dynamic expression pattern. Mutants lacking the catalytic domain of PkcA (pkcA−) did not exhibit tip dominance. A striking phenotype of pkcA− was the formation of an aggregate with a central hollow, and aggregates later fragmented to form small mounds, each becoming a fruiting body. Optical density wave patterns of cAMP in the late aggregates showed several cAMP wave generation centers. We attribute these defects in pkcA− to impaired cAMP signaling, altered cell motility and decreased expression of the cell adhesion molecules – CadA and CsaA. pkcA− slugs showed ectopic expression of ecmA in the prespore region. Further, the use of a PKC-specific inhibitor, GF109203X that inhibits the activity of catalytic domain phenocopied pkcA−. PMID:26183108

Msx2 Prevents Stratified Squamous Epithelium Formation in the Enamel Organ.

PubMed

Nakatomi, M; Ida-Yonemochi, H; Nakatomi, C; Saito, K; Kenmotsu, S; Maas, R L; Ohshima, H

2018-06-01

Tooth enamel is manufactured by the inner enamel epithelium of the multilayered enamel organ. Msx2 loss-of-function mutation in a mouse model causes an abnormal accumulation of epithelial cells in the enamel organ, but the underlying mechanism by which Msx2 regulates amelogenesis is poorly understood. We therefore performed detailed histological and molecular analyses of Msx2 null mice. Msx2 null ameloblasts and stratum intermedium (SI) cells differentiated normally in the early stages of amelogenesis. However, during subsequent developmental stages, the outer enamel epithelium (OEE) became highly proliferative and transformed into a keratinized stratified squamous epithelium that ectopically expressed stratified squamous epithelium markers, including Heat shock protein 25, Loricrin, and Keratin 10. Moreover, expression of hair follicle-specific keratin genes such as Keratin 26 and Keratin 73 was upregulated in the enamel organ of Msx2 mutants. With the accumulation of keratin in the stellate reticulum (SR) region and subsequent odontogenic cyst formation, SI cells gradually lost the ability to differentiate, and the expression of Sox2 and Notch1 was downregulated, leading to ameloblast depolarization. As a consequence, the organization of the Msx2 mutant enamel organ became disturbed and enamel failed to form in the normal location. Instead, there was ectopic mineralization that likely occurred within the SR. In summary, we show that during amelogenesis, Msx2 executes a bipartite function, repressing the transformation of OEE into a keratinized stratified squamous epithelium while simultaneously promoting the development of a properly differentiated enamel organ competent for enamel formation.

CRISPR/Cas9 Mediated GFP Knock-in at the MAP1LC3B Locus in 293FT Cells Is Better for Bona Fide Monitoring Cellular Autophagy.

PubMed

Wu, Zhiqiang; Zhao, Jinlin; Qiu, Minghan; Mi, Zeyun; Meng, Maobin; Guo, Yu; Wang, Hui; Yuan, Zhiyong

2018-04-19

Accurately identifying and quantifying cellular autophagy is very important as the significance of autophagy in physiological and pathological processes becomes increasingly evident. Ectopically expressed fluorescent-tagged microtubule-associated protein light chain 3B (MAP1LC3B, LC3) is the most widely used reporter for monitoring autophagy activity thus far. However, this approach ignores the influence of constitutively overexpressed LC3 on autophagy itself and autophagy-related processes and its accuracy in indicating autophagy is questionable. Here, we generated a knock-in GFP-LC3 reporter via the CRISPR/Cas9 system in 293FT cells to add GFP to the N-terminal of and in frame with endogenous LC3. We proved that this knock-in GFP-LC3 was expressed at biological level driven by the endogenous transcriptional regulatory elements as the wild type alleles. Compared with the ectopically expressed GFP-LC3, the endogenous knock-in reporter exhibited much higher sensitivity and signal-to-noise ratio of GFP-LC3 puncta upon the induction or inhibition of autophagy at certain step for monitoring autophagy activity. Thus, according to the previous reported concerning and the results presented here, we suggest that this knock-in GFP-LC3 reporter is better for bona fide monitoring cellular autophagy and should be employed for further study of autophagy in vitro and in vivo. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Effect of Hua Yu Xiao Zheng decoction on the expression levels of vascular endothelial growth factor and angiopoietin-2 in rats with endometriosis.

PubMed

Chen, Zhen-Zhen; Gong, Xin

2017-12-01

The aims of the present study were to investigate the effects of a traditional Chinese medicine, Hua Yu Xiao Zheng (HYXZ) decoction, on surgically induced endometriosis in a rat model and to determine the possible underlying regulatory mechanisms. A total of 108 female Sprague-Dawley rats were divided into the control group (n=12) and endometriosis group (EM group; n=96), in which endometriosis was surgically induced in model rats by autotransplantation of endometrial tissues and 72 rats survived. After 3 weeks, the EM model rats were randomly divided into four subgroups (n=18), including the untreated model group, and three groups administered 7, 14 or 21 g/kg HYXZ decoction. Following 28 days of treatment, the associated proteins and genes of ectopic endometrial tissues were analyzed using immunohistochemistry, western blotting and quantitative polymerase chain reaction to investigate the underlying mechanisms. Compared with the model group, the size of the endometriotic implants decreased significantly in the HYXZ-treated groups. Furthermore, the expression levels of vascular endothelial growth factor (VEGF) and angiopoietin-2 (Ang-2) were significantly decreased in HYXZ-treated groups compared with the model group. These results indicate that HYXZ affected the inhibition of angiogenesis and decreased the endometriotic implant volumes and histopathological scores. The effectiveness of HYXZ may be partially attributed to the decrease of VEGF and Ang-2 expression levels in the ectopic endometrium.

Protein-anchoring therapy to target extracellular matrix proteins to their physiological destinations.

PubMed

Ito, Mikako; Ohno, Kinji

2018-02-20

Endplate acetylcholinesterase (AChE) deficiency is a form of congenital myasthenic syndrome (CMS) caused by mutations in COLQ, which encodes collagen Q (ColQ). ColQ is an extracellular matrix (ECM) protein that anchors AChE to the synaptic basal lamina. Biglycan, encoded by BGN, is another ECM protein that binds to the dystrophin-associated protein complex (DAPC) on skeletal muscle, which links the actin cytoskeleton and ECM proteins to stabilize the sarcolemma during repeated muscle contractions. Upregulation of biglycan stabilizes the DPAC. Gene therapy can potentially ameliorate any disease that can be recapitulated in cultured cells. However, the difficulty of tissue-specific and developmental stage-specific regulated expression of transgenes, as well as the difficulty of introducing a transgene into all cells in a specific tissue, prevents us from successfully applying gene therapy to many human diseases. In contrast to intracellular proteins, an ECM protein is anchored to the target tissue via its specific binding affinity for protein(s) expressed on the cell surface within the target tissue. Exploiting this unique feature of ECM proteins, we developed protein-anchoring therapy in which a transgene product expressed even in remote tissues can be delivered and anchored to a target tissue using specific binding signals. We demonstrate the application of protein-anchoring therapy to two disease models. First, intravenous administration of adeno-associated virus (AAV) serotype 8-COLQ to Colq-deficient mice, resulting in specific anchoring of ectopically expressed ColQ-AChE at the NMJ, markedly improved motor functions, synaptic transmission, and the ultrastructure of the neuromuscular junction (NMJ). In the second example, Mdx mice, a model for Duchenne muscular dystrophy, were intravenously injected with AAV8-BGN. The treatment ameliorated motor deficits, mitigated muscle histopathologies, decreased plasma creatine kinase activities, and upregulated expression of utrophin and DAPC component proteins. We propose that protein-anchoring therapy could be applied to hereditary/acquired defects in ECM and secreted proteins, as well as therapeutic overexpression of such factors. Copyright © 2017 International Society of Matrix Biology. Published by Elsevier B.V. All rights reserved.

A plasma membrane sucrose-binding protein that mediates sucrose uptake shares structural and sequence similarity with seed storage proteins but remains functionally distinct.

PubMed

Overvoorde, P J; Chao, W S; Grimes, H D

1997-06-20

Photoaffinity labeling of a soybean cotyledon membrane fraction identified a sucrose-binding protein (SBP). Subsequent studies have shown that the SBP is a unique plasma membrane protein that mediates the linear uptake of sucrose in the presence of up to 30 mM external sucrose when ectopically expressed in yeast. Analysis of the SBP-deduced amino acid sequence indicates it lacks sequence similarity with other known transport proteins. Data presented here, however, indicate that the SBP shares significant sequence and structural homology with the vicilin-like seed storage proteins that organize into homotrimers. These similarities include a repeated sequence that forms the basis of the reiterated domain structure characteristic of the vicilin-like protein family. In addition, analytical ultracentrifugation and nonreducing SDS-polyacrylamide gel electrophoresis demonstrate that the SBP appears to be organized into oligomeric complexes with a Mr indicative of the existence of SBP homotrimers and homodimers. The structural similarity shared by the SBP and vicilin-like proteins provides a novel framework to explore the mechanistic basis of SBP-mediated sucrose uptake. Expression of the maize Glb protein (a vicilin-like protein closely related to the SBP) in yeast demonstrates that a closely related vicilin-like protein is unable to mediate sucrose uptake. Thus, despite sequence and structural similarities shared by the SBP and the vicilin-like protein family, the SBP is functionally divergent from other members of this group.

Dexamethasone Enhances Osteogenic Differentiation of Bone Marrow- and Muscle-Derived Stromal Cells and Augments Ectopic Bone Formation Induced by Bone Morphogenetic Protein-2

PubMed Central

Yuasa, Masato; Yamada, Tsuyoshi; Taniyama, Takashi; Masaoka, Tomokazu; Xuetao, Wei; Yoshii, Toshitaka; Horie, Masaki; Yasuda, Hiroaki; Uemura, Toshimasa; Okawa, Atsushi; Sotome, Shinichi

2015-01-01

We evaluated whether dexamethasone augments the osteogenic capability of bone marrow-derived stromal cells (BMSCs) and muscle tissue-derived stromal cells (MuSCs), both of which are thought to contribute to ectopic bone formation induced by bone morphogenetic protein-2 (BMP-2), and determined the underlying mechanisms. Rat BMSCs and MuSCs were cultured in growth media with or without 10-7 M dexamethasone and then differentiated under osteogenic conditions with dexamethasone and BMP-2. The effects of dexamethasone on cell proliferation and osteogenic differentiation, and also on ectopic bone formation induced by BMP-2, were analyzed. Dexamethasone affected not only the proliferation rate but also the subpopulation composition of BMSCs and MuSCs, and subsequently augmented their osteogenic capacity during osteogenic differentiation. During osteogenic induction by BMP-2, dexamethasone also markedly affected cell proliferation in both BMSCs and MuSCs. In an in vivo ectopic bone formation model, bone formation in muscle-implanted scaffolds containing dexamethasone and BMP-2 was more than two fold higher than that in scaffolds containing BMP-2 alone. Our results suggest that dexamethasone potently enhances the osteogenic capability of BMP-2 and may thus decrease the quantity of BMP-2 required for clinical application, thereby reducing the complications caused by excessive doses of BMP-2. Highlights: 1. Dexamethasone induced selective proliferation of bone marrow- and muscle-derived cells with higher differentiation potential. 2. Dexamethasone enhanced the osteogenic capability of bone marrow- and muscle-derived cells by altering the subpopulation composition. 3. Dexamethasone augmented ectopic bone formation induced by bone morphogenetic protein-2. PMID:25659106

Ectopic expression of Msx-2 in posterior limb bud mesoderm impairs limb morphogenesis while inducing BMP-4 expression, inhibiting cell proliferation, and promoting apoptosis.

PubMed

Ferrari, D; Lichtler, A C; Pan, Z Z; Dealy, C N; Upholt, W B; Kosher, R A

1998-05-01

During early stages of chick limb development, the homeobox-containing gene Msx-2 is expressed in the mesoderm at the anterior margin of the limb bud and in a discrete group of mesodermal cells at the midproximal posterior margin. These domains of Msx-2 expression roughly demarcate the anterior and posterior boundaries of the progress zone, the highly proliferating posterior mesodermal cells underneath the apical ectodermal ridge (AER) that give rise to the skeletal elements of the limb and associated structures. Later in development as the AER loses its activity, Msx-2 expression expands into the distal mesoderm and subsequently into the interdigital mesenchyme which demarcates the developing digits. The domains of Msx-2 expression exhibit considerably less proliferation than the cells of the progress zone and also encompass several regions of programmed cell death including the anterior and posterior necrotic zones and interdigital mesenchyme. We have thus suggested that Msx-2 may be in a regulatory network that delimits the progress zone by suppressing the morphogenesis of the regions of the limb mesoderm in which it is highly expressed. In the present study we show that ectopic expression of Msx-2 via a retroviral expression vector in the posterior mesoderm of the progress zone from the time of initial formation of the limb bud severely impairs limb morphogenesis. Msx-2-infected limbs are typically very narrow along the anteroposterior axis, are occasionally truncated, and exhibit alterations in the pattern of formation of skeletal elements, indicating that as a consequence of ectopic Msx-2 expression the morphogenesis of large portions of the posterior mesoderm has been suppressed. We further show that Msx-2 impairs limb morphogenesis by reducing cell proliferation and promoting apoptosis in the regions of the posterior mesoderm in which it is ectopically expressed. The domains of ectopic Msx-2 expression in the posterior mesoderm also exhibit ectopic expression of BMP-4, a secreted signaling molecule that is coexpressed with Msx-2 during normal limb development in the anterior limb mesoderm, the posterior necrotic zone, and interdigital mesenchyme. This indicates that Msx-2 regulates BMP-4 expression and that the suppressive effects of Msx-2 on limb morphogenesis might be mediated in part by BMP-4. These studies indicate that during normal limb development Msx-2 is a key component of a regulatory network that delimits the boundaries of the progress zone by suppressing the morphogenesis of the regions of the limb mesoderm in which it is highly expressed, thus restricting the outgrowth and formation of skeletal elements and associated structures to the progress zone. We also report that rather large numbers of apoptotic cells as well as proliferating cells are present throughout the AER during all stages of normal limb development we have examined, indicating that many of the cells of the AER are continuously undergoing programmed cell death at the same time that new AER cells are being generated by cell proliferation. Thus, a balance between cell proliferation and programmed cell death may play a very important role in maintaining the activity of the AER. Copyright 1998 Academic Press.

PRDM1 expression via human parvovirus B19 infection plays a role in the pathogenesis of Hashimoto thyroiditis.

PubMed

Wang, Lu; Zhang, Wei-Ping; Yao, Li; Zhang, Wei; Zhu, Jin; Zhang, Wei-Chen; Zhang, Yue-Hua; Wang, Zhe; Yan, Qing-Guo; Guo, Ying; Fan, Lin-Ni; Liu, Yi-Xiong; Huang, Gao-Sheng

2015-12-01

Ectopic lymphoid follicle infiltration is a key event in Hashimoto thyroiditis (HT). Positive regulatory domain zinc finger protein 1 (PRDM1), which is induced by antigen stimulation, can regulate all lymphocyte lineages. Several groups independently demonstrated that human parvovirus B19 (PVB19) is closely associated with HT. Hence, we determined whether PRDM1 is expressed in HT thyroid tissue and whether there is any correlation between PRDM1 expression and PVB19 in the pathogenesis of HT. We detected PRDM1 expression in HT (n = 86), normal thyroid tissue (n = 30), and nontoxic nodular goiter (n = 20) samples using immunohistochemistry. We also detected PVB19 protein in HT samples in a double-blind manner and analyzed the correlation between the 2 proteins using immunofluorescence confocal detection and coimmunoprecipitation. Furthermore, we detected changes of the expression levels of PRDM1 and PVB19 in transfected primary thyroid follicular epithelial cells using real-time quantitative polymerase chain reaction. We found that PRDM1 protein is significantly highly expressed in the injured follicular epithelial cells in HT (83/86 cases) than in normal thyroid cells (0/30 cases) or in nontoxic nodular goiter cells (0/20 cases) (P < .001). In HT, the PRDM1 expression pattern was the same as that of PVB19, whereas PRDM1 and PVB19 were coexistent in the involved epithelial cells. Statistical analysis showed a significant correlation between PRDM1 and PVB19 (P < .001). In addition, primary thyroid epithelial cells also showed PRDM1 up-regulation after PVB19 NS1 transfection. Our findings suggest a previously unrecognized role of PRDM1 and PVB19 in the pathogenesis of HT. Copyright © 2015 Elsevier Inc. All rights reserved.

Vaccinia-related kinase 3 (VRK3) sets the circadian period and amplitude by affecting the subcellular localization of clock proteins in mammalian cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Park, Nayoung; Department of Brain Science, Ajou University School of Medicine, 164 Worldcup-ro, Yeongtong-gu, Suwon, Kyunggi-do, 16499; Song, Jieun

In the eukaryotic circadian clock machinery, negative feedback repression of CLOCK (CLK) and BMAL1 transcriptional activity by PERIOD (PER) and CRYPTOCHROME (CRY) underlies the basis for 24 h rhythmic gene expression. Thus, precise regulation of the time-dependent nuclear entry of circadian repressors is crucial to generating normal circadian rhythms. Here, we sought to identify novel kinase(s) that regulate nuclear entry of mammalian CRY1 (mCRY1) with an unbiased screening using red fluorescent protein (RFP)-tagged human kinome expression plasmids in mammalian cells. Transient expression of human vaccinia-related kinase 3 (hVRK3) reduced the nuclear presence of mCRY1. hVRK3 expression also induced alterations in themore » subcellular localization of other core clock proteins, including mCRY2, mPER2, and BMAL1. In contrast, the subcellular localization of mCLK was not changed. Given that singly expressed mCLK mostly resides in the cytoplasm and that nuclear localization sequence (NLS) mutation of hVRK3 attenuated the effect of hVRK3 co-expression on subcellular localization, ectopically expressed hVRK3 presumably reduces the retention of proteins in the nucleus. Finally, downregulation of hvrk3 using siRNA reduced the amplitude and lengthened the period of the cellular bioluminescence rhythm. Taken together, these data suggest that VRK3 plays a role in setting the amplitude and period length of circadian rhythms in mammalian cells. - Highlights: • Screening was performed to identify kinases that regulate CRY1 subcellular localization. • VRK3 alters the subcellular localization of CRY1, CRY2, PER2, and BMAL1. • VRK3 knock-down alters the circadian bioluminescence rhythm in mammalian cells.« less

Selective Expression of an Endogenous Inhibitor of FAK Regulates Proliferation and Migration of Vascular Smooth Muscle Cells

PubMed Central

Taylor, Joan M.; Mack, Christopher P.; Nolan, Kate; Regan, Christopher P.; Owens, Gary K.; Parsons, J. Thomas

2001-01-01

Extracellular matrix signaling via integrin receptors is important for smooth muscle cell (SMC) differentiation during vasculogenesis and for phenotypic modulation of SMCs during atherosclerosis. We previously reported that the noncatalytic carboxyl-terminal protein binding domain of focal adhesion kinase (FAK) is expressed as a separate protein termed FAK-related nonkinase (FRNK) and that ectopic expression of FRNK can attenuate FAK activity and integrin-dependent signaling (A. Richardson and J. T. Parsons, Nature 380:538–540, 1996). Herein we report that in contrast to FAK, which is expressed ubiquitously, FRNK is expressed selectively in SMCs, with particularly high levels observed in conduit blood vessels. FRNK expression was low during embryonic development, was significantly upregulated in the postnatal period, and returned to low but detectable levels in adult tissues. FRNK expression was also dramatically upregulated following balloon-induced carotid artery injury. In cultured rat aortic smooth muscle cells, overexpression of FRNK attenuated platelet-derived growth factor (PDGF)-BB-induced migration and also dramatically inhibited [3H]thymidine incorporation upon stimulation with PDGF-BB or 10% serum. These effects were concomitant with a reduction in SMC proliferation. Taken together, these data indicate that FRNK acts as an endogenous inhibitor of FAK signaling in SMCs. Furthermore, increased FRNK expression following vascular injury or during development may alter the SMC phenotype by negatively regulating proliferative and migratory signals. PMID:11238893

Expression of grape ACS1 in tomato decreases ethylene and alters the balance between auxin and ethylene during shoot and root formation.

PubMed

Ye, Xia; Fu, Mengmeng; Liu, Yu; An, Dongliang; Zheng, Xianbo; Tan, Bin; Li, Jidong; Cheng, Jun; Wang, Wei; Feng, Jiancan

2018-05-04

Ethylene plays an important role in the grape rachis, where its production can be 10 times higher than in the berry. VvACS1 is the only rachis-specific ACC synthase (ACS) gene, and its expression is coincident with ethylene production in the rachis of Vitis vinifera 'Thompson seedless'. VvACS1 was cloned and ectopically expressed in tomato (Solanum lycopersicum 'Moneymaker'). Lateral buds were increased in two- or four-week-old 35s∷VvACS1 transgenic tomato plants after transplanting. Compared with wild-type (WT) plants, the transgenic tomato plants showed higher expression of the VvACS1 gene in the flowers, leaves, rachis, and fruits. There was no obvious difference of ACS activity in the fruit of tomato, and only increased ACS activity in the rachis of tomato. Ethylene production was decreased in flowers, leaves, and fruits (seven weeks after full bloom), while the relative expression of endogenous tomato ACS1 and ACS6 genes was not down-regulated by the ectopic expression of VvACS1. These results imply that post-transcriptional or post-translational regulation of ACS may occur, resulting in lower ethylene production in the transgenic tomato plants. Moreover, expression of VvACS1 in tomato resulted in decreased auxin and increased zeatin contents in the lateral buds, as well as reduced or delayed formation of adventitious roots in lateral bud cuttings. RNA-Seq and qRT-PCR analyses of rooted lateral bud cuttings indicated that the relative expression levels of the genes for zeatin O-glucosyltransferase-like, auxin repressed/dormancy-associated protein, and ERF transcription factors were higher in transgenic tomatoes than in WT, suggesting that ethylene may regulate auxin transport and distribution in shoots and that adventitious root formation employs coordination between auxin and ethylene. Copyright © 2018 Elsevier GmbH. All rights reserved.

Overexpression of caspase 7 is ERα dependent to affect proliferation and cell growth in breast cancer cells by targeting p21(Cip).

PubMed

Chaudhary, S; Madhukrishna, B; Adhya, A K; Keshari, S; Mishra, S K

2016-04-18

Caspase 7 (CASP7) expression has important function during cell cycle progression and cell growth in certain cancer cells and is also involved in the development and differentiation of dental tissues. However, the function of CASP7 in breast cancer cells is unclear. The aim of this study was to analyze the expression of CASP7 in breast carcinoma patients and determine the role of CASP7 in regulating tumorigenicity in breast cancer cells. In this study, we show that the CASP7 expression is high in breast carcinoma tissues compared with normal counterpart. The ectopic expression of CASP7 is significantly associated with ERα expression status and persistently elevated in different stages of the breast tumor grades. High level of CASP7 expression showed better prognosis in breast cancer patients with systemic endocrine therapy as observed from Kaplan-Meier analysis. S3 and S4, estrogen responsive element (ERE) in the CASP7 promoter, is important for estrogen-ERα-mediated CASP7 overexpression. Increased recruitment of p300, acetylated H3 and pol II in the ERE region of CASP7 promoter is observed after hormone stimulation. Ectopic expression of CASP7 in breast cancer cells results in cell growth and proliferation inhibition via p21(Cip) reduction, whereas small interfering RNA (siRNA) mediated reduction of CASP7 rescued p21(Cip) levels. We also show that pro- and active forms of CASP7 is located in the nucleus apart from cytoplasmic region of breast cancer cells. The proliferation and growth of breast cancer cells is significantly reduced by broad-spectrum peptide inhibitors and siRNA of CASP7. Taken together, our findings show that CASP7 is aberrantly expressed in breast cancer and contributes to cell growth and proliferation by downregulating p21(Cip) protein, suggesting that targeting CASP7-positive breast cancer could be one of the potential therapeutic strategies.

Microarray analyses reveal distinct roles for Rel proteins in the Drosophila immune response

PubMed Central

Pal, Subhamoy; Wu, Junlin; Wu, Louisa P.

2007-01-01

The NF-κB group of transcription factors play an important role in mediating immune responses in organisms as diverse as insects and mammals. The fruit fly Drosophila melanogaster express three closely related NF-κB-like transcription factors: Dorsal, Dif, and Relish. To study their roles in vivo, we used microarrays to determine the effect of null mutations in individual Rel transcription factors on larval immune gene expression. Of the 188 genes that were significantly up-regulated in wildtype larvae upon bacterial challenge, overlapping but distinct groups of genes were affected in the Rel mutants. We also ectopically expressed Dorsal or Dif and used cDNA microarrays to determine the genes that were up-regulated in the presence of these transcription factors. This expression was sufficient to drive expression of some immune genes, suggesting redundancy in the regulation of these genes. Combining this data, we also identified novel genes that may be specific targets of Dif. PMID:17537510

crm-1 facilitates BMP signaling to control body size in Caenorhabditis elegans.

PubMed

Fung, Wong Yan; Fat, Ko Frankie Chi; Eng, Cheah Kathryn Song; Lau, Chow King

2007-11-01

We have identified in Caenorhabditis elegans a homologue of the vertebrate Crim1, crm-1, which encodes a putative transmembrane protein with multiple cysteine-rich (CR) domains known to have bone morphogenetic proteins (BMPs) binding activity. Using the body morphology of C. elegans as an indicator, we showed that attenuation of crm-1 activity leads to a small body phenotype reminiscent of that of BMP pathway mutants. We showed that the crm-1 loss-of-function phenotype can be rescued by constitutive supply of sma-4 activity. crm-1 can enhance BMP signaling and this activity is dependent on the presence of the DBL-1 ligand and its receptors. crm-1 is expressed in neurons at the ventral nerve cord, where the DBL-1 ligand is produced. However, ectopic expression experiments reveal that crm-1 gene products act outside the DBL-1 producing cells and function non-autonomously to facilitate dbl/sma pathway signaling to control body size.

TRIM67 Protein Negatively Regulates Ras Activity through Degradation of 80K-H and Induces Neuritogenesis*

PubMed Central

Yaguchi, Hiroaki; Okumura, Fumihiko; Takahashi, Hidehisa; Kano, Takahiro; Kameda, Hiroyuki; Uchigashima, Motokazu; Tanaka, Shinya; Watanabe, Masahiko; Sasaki, Hidenao; Hatakeyama, Shigetsugu

2012-01-01

Tripartite motif (TRIM)-containing proteins, which are defined by the presence of a common domain structure composed of a RING finger, one or two B-box motifs and a coiled-coil motif, are involved in many biological processes including innate immunity, viral infection, carcinogenesis, and development. Here we show that TRIM67, which has a TRIM motif, an FN3 domain and a SPRY domain, is highly expressed in the cerebellum and that TRIM67 interacts with PRG-1 and 80K-H, which is involved in the Ras-mediated signaling pathway. Ectopic expression of TRIM67 results in degradation of endogenous 80K-H and attenuation of cell proliferation and enhances neuritogenesis in the neuroblastoma cell line N1E-115. Furthermore, morphological and biological changes caused by knockdown of 80K-H are similar to those observed by overexpression of TRIM67. These findings suggest that TRIM67 regulates Ras signaling via degradation of 80K-H, leading to neural differentiation including neuritogenesis. PMID:22337885

TRIM67 protein negatively regulates Ras activity through degradation of 80K-H and induces neuritogenesis.

PubMed

Yaguchi, Hiroaki; Okumura, Fumihiko; Takahashi, Hidehisa; Kano, Takahiro; Kameda, Hiroyuki; Uchigashima, Motokazu; Tanaka, Shinya; Watanabe, Masahiko; Sasaki, Hidenao; Hatakeyama, Shigetsugu

2012-04-06

Tripartite motif (TRIM)-containing proteins, which are defined by the presence of a common domain structure composed of a RING finger, one or two B-box motifs and a coiled-coil motif, are involved in many biological processes including innate immunity, viral infection, carcinogenesis, and development. Here we show that TRIM67, which has a TRIM motif, an FN3 domain and a SPRY domain, is highly expressed in the cerebellum and that TRIM67 interacts with PRG-1 and 80K-H, which is involved in the Ras-mediated signaling pathway. Ectopic expression of TRIM67 results in degradation of endogenous 80K-H and attenuation of cell proliferation and enhances neuritogenesis in the neuroblastoma cell line N1E-115. Furthermore, morphological and biological changes caused by knockdown of 80K-H are similar to those observed by overexpression of TRIM67. These findings suggest that TRIM67 regulates Ras signaling via degradation of 80K-H, leading to neural differentiation including neuritogenesis.

An Acidic PATHOGENESIS-RELATED1 Gene of Oryza grandiglumis is Involved in Disease Resistance Response Against Bacterial Infection

PubMed Central

Shin, Sang Hyun; Pak, Jung-Hun; Kim, Mi Jin; Kim, Hye Jeong; Oh, Ju Sung; Choi, Hong Kyu; Jung, Ho Won; Chung, Young Soo

2014-01-01

Wild rice, Oryza grandiglumis shows hyper-resistance response to pathogen infection. In order to identify genes necessary for defense response in plants, we have carried out a subtractive hybridization coupled with a cDNA macroarray. An acidic PATHOGENESIS-RELATED1 (PR1) gene of the wild rice is highly identical to the acidic PR1 genes of different plant species. The OgPR1a cDNA has an apparent single open reading frame with a predicted molecular mass 40,621 Da and an isoelectic point of 5.14. Both in silico analysis and a transient expression assay in onion epidermal cells revealed that the OgPR1a protein could be localized in intercellular space in plants. The OgPR1a mRNA was strongly transcribed by the exogenous treatment with ethylene and jasmonic acid as well as protein phosphatase inhibitors. Additionally, ectopic expression of the OgPR1a conferred disease resistance on Arabidopsis to the bacterial and fungal infections. PMID:25289005

Regulation of Synaptic Structure by the Ubiquitin C-terminal Hydrolase UCH-L1

PubMed Central

Cartier, Anna E.; Djakovic, Stevan N.; Salehi, Afshin; Wilson, Scott M.; Masliah, Eliezer; Patrick, Gentry N.

2009-01-01

UCH-L1 is a de-ubiquitinating enzyme that is selectively and abundantly expressed in the brain, and its activity is required for normal synaptic function. Here, we show that UCH-L1 functions in maintaining normal synaptic structure in hippocampal neurons. We have found that UCH-L1 activity is rapidly up-regulated by NMDA receptor activation which leads to an increase in the levels of free monomeric ubiquitin. Conversely, pharmacological inhibition of UCH-L1 significantly reduces monomeric ubiquitin levels and causes dramatic alterations in synaptic protein distribution and spine morphology. Inhibition of UCH-L1 activity increases spine size while decreasing spine density. Furthermore, there is a concomitant increase in the size of pre and postsynaptic protein clusters. Interestingly, however, ectopic expression of ubiquitin restores normal synaptic structure in UCH-L1 inhibited neurons. These findings point to a significant role of UCH-L1 in synaptic remodeling most likely by modulating free monomeric ubiquitin levels in an activity-dependent manner. PMID:19535597

Regulation of synaptic structure by ubiquitin C-terminal hydrolase L1.

PubMed

Cartier, Anna E; Djakovic, Stevan N; Salehi, Afshin; Wilson, Scott M; Masliah, Eliezer; Patrick, Gentry N

2009-06-17

Ubiquitin C-terminal hydrolase L1 (UCH-L1) is a deubiquitinating enzyme that is selectively and abundantly expressed in the brain, and its activity is required for normal synaptic function. Here, we show that UCH-L1 functions in maintaining normal synaptic structure in hippocampal neurons. We found that UCH-L1 activity is rapidly upregulated by NMDA receptor activation, which leads to an increase in the levels of free monomeric ubiquitin. Conversely, pharmacological inhibition of UCH-L1 significantly reduces monomeric ubiquitin levels and causes dramatic alterations in synaptic protein distribution and spine morphology. Inhibition of UCH-L1 activity increases spine size while decreasing spine density. Furthermore, there is a concomitant increase in the size of presynaptic and postsynaptic protein clusters. Interestingly, however, ectopic expression of ubiquitin restores normal synaptic structure in UCH-L1-inhibited neurons. These findings point to a significant role of UCH-L1 in synaptic remodeling, most likely by modulating free monomeric ubiquitin levels in an activity-dependent manner.

Fatty Acid-Binding Protein 4 (FABP4): Pathophysiological Insights and Potent Clinical Biomarker of Metabolic and Cardiovascular Diseases

PubMed Central

Furuhashi, Masato; Saitoh, Shigeyuki; Shimamoto, Kazuaki; Miura, Tetsuji

2014-01-01

Over the past decade, evidences of an integration of metabolic and inflammatory pathways, referred to as metaflammation in several aspects of metabolic syndrome, have been accumulating. Fatty acid-binding protein 4 (FABP4), also known as adipocyte FABP (A-FABP) or aP2, is mainly expressed in adipocytes and macrophages and plays an important role in the development of insulin resistance and atherosclerosis in relation to metaflammation. Despite lack of a typical secretory signal peptide, FABP4 has been shown to be released from adipocytes in a non-classical pathway associated with lipolysis, possibly acting as an adipokine. Elevation of circulating FABP4 levels is associated with obesity, insulin resistance, diabetes mellitus, hypertension, cardiac dysfunction, atherosclerosis, and cardiovascular events. Furthermore, ectopic expression and function of FABP4 in several types of cells and tissues have been recently demonstrated. Here, we discuss both the significant role of FABP4 in pathophysiological insights and its usefulness as a biomarker of metabolic and cardiovascular diseases. PMID:25674026

Fatty Acid-Binding Protein 4 (FABP4): Pathophysiological Insights and Potent Clinical Biomarker of Metabolic and Cardiovascular Diseases.

PubMed

Furuhashi, Masato; Saitoh, Shigeyuki; Shimamoto, Kazuaki; Miura, Tetsuji

2014-01-01

Over the past decade, evidences of an integration of metabolic and inflammatory pathways, referred to as metaflammation in several aspects of metabolic syndrome, have been accumulating. Fatty acid-binding protein 4 (FABP4), also known as adipocyte FABP (A-FABP) or aP2, is mainly expressed in adipocytes and macrophages and plays an important role in the development of insulin resistance and atherosclerosis in relation to metaflammation. Despite lack of a typical secretory signal peptide, FABP4 has been shown to be released from adipocytes in a non-classical pathway associated with lipolysis, possibly acting as an adipokine. Elevation of circulating FABP4 levels is associated with obesity, insulin resistance, diabetes mellitus, hypertension, cardiac dysfunction, atherosclerosis, and cardiovascular events. Furthermore, ectopic expression and function of FABP4 in several types of cells and tissues have been recently demonstrated. Here, we discuss both the significant role of FABP4 in pathophysiological insights and its usefulness as a biomarker of metabolic and cardiovascular diseases.

EGFR-targeted granzyme B expressed in NK cells enhances natural cytotoxicity and mediates specific killing of tumor cells.

PubMed

Oberoi, Pranav; Jabulowsky, Robert A; Bähr-Mahmud, Hayat; Wels, Winfried S

2013-01-01

Natural killer (NK) cells are highly specialized effectors of the innate immune system that hold promise for adoptive cancer immunotherapy. Their cell killing activity is primarily mediated by the pro-apoptotic serine protease granzyme B (GrB), which enters targets cells with the help of the pore-forming protein perforin. We investigated expression of a chimeric GrB fusion protein in NK cells as a means to augment their antitumoral activity. For selective targeting to tumor cells, we fused the epidermal growth factor receptor (EGFR) peptide ligand transforming growth factor α (TGFα) to human pre-pro-GrB. Established human NKL natural killer cells transduced with a lentiviral vector expressed this GrB-TGFα (GrB-T) molecule in amounts comparable to endogenous wildtype GrB. Activation of the genetically modified NK cells by cognate target cells resulted in the release of GrB-T together with endogenous granzymes and perforin, which augmented the effector cells' natural cytotoxicity against NK-sensitive tumor cells. Likewise, GrB-T was released into the extracellular space upon induction of degranulation with PMA and ionomycin. Secreted GrB-T fusion protein displayed specific binding to EGFR-overexpressing tumor cells, enzymatic activity, and selective target cell killing in the presence of an endosomolytic activity. Our data demonstrate that ectopic expression of a targeted GrB fusion protein in NK cells is feasible and can enhance antitumoral activity of the effector cells.

Epigenetic repression of regulator of G-protein signaling 2 promotes androgen-independent prostate cancer cell growth.

PubMed

Wolff, Dennis W; Xie, Yan; Deng, Caishu; Gatalica, Zoran; Yang, Mingjie; Wang, Bo; Wang, Jincheng; Lin, Ming-Fong; Abel, Peter W; Tu, Yaping

2012-04-01

G-protein-coupled receptor (GPCR)-stimulated androgen-independent activation of androgen receptor (AR) contributes to acquisition of a hormone-refractory phenotype by prostate cancer. We previously reported that regulator of G-protein signaling (RGS) 2, an inhibitor of GPCRs, inhibits androgen-independent AR activation (Cao et al., Oncogene 2006;25:3719-34). Here, we show reduced RGS2 protein expression in human prostate cancer specimens compared to adjacent normal or hyperplastic tissue. Methylation-specific PCR analysis and bisulfite sequencing indicated that methylation of the CpG island in the RGS2 gene promoter correlated with RGS2 downregulation in prostate cancer. In vitro methylation of this promoter suppressed reporter gene expression in transient transfection studies, whereas reversal of this promoter methylation with 5-aza-2'-deoxycytidine (5-Aza-dC) induced RGS2 reexpression in androgen-independent prostate cancer cells and inhibited their growth under androgen-deficient conditions. Interestingly, the inhibitory effect of 5-Aza-dC was significantly reduced by an RGS2-targeted short hairpin RNA, indicating that reexpressed RGS2 contributed to this growth inhibition. Restoration of RGS2 levels by ectopic expression in androgen-independent prostate cancer cells suppressed growth of xenografts in castrated mice. Thus, RGS2 promoter hypermethylation represses its expression and unmasks a latent pathway for AR transactivation in prostate cancer cells. Targeting this reversible process may provide a new strategy for suppressing prostate cancer progression by reestablishing its androgen sensitivity. Copyright © 2011 UICC.

The pituitary V3 vasopressin receptor and the corticotroph phenotype in ectopic ACTH syndrome.

PubMed Central

de Keyzer, Y; Lenne, F; Auzan, C; Jégou, S; René, P; Vaudry, H; Kuhn, J M; Luton, J P; Clauser, E; Bertagna, X

1996-01-01

Ectopic ACTH secretion occurs in highly differentiated and rather indolent tumors like bronchial carcinoids or, in contrast, in various types of aggressive and poorly differentiated neuroendocrine tumors. We explored this phenomenon using the recently cloned human pituitary V3 vasopressin receptor as an alternate molecular marker of the corticotroph phenotype. Expression of V3 receptor, corticotrophin releasing hormone (CRH) receptor, and proopiomelanocortin (POMC) genes was examined in tumors of pituitary and nonpituitary origin. A comparative RT-PCR approach revealed signals for both V3 receptor and CHR receptor mRNAs in 17 of 18 ACTH-secreting pituitary adenomas, and 6 of 6 normal pituitaries; in six growth hormone- or prolactin-secreting adenomas, a very faint V3 receptor signal was observed in three cases, and CRH receptor signal was undetected in all. Six of eight bronchial carcinoids responsible for the ectopic ACTH syndrome had both POMC and V3 receptor signals as high as those in ACTH-secreting pituitary adenomas; in contrast, no POMC signal and only a very faint V3 receptor signal were detected in six of eight nonsecreting bronchial carcinoids. Northern blot analysis showed V3 receptor mRNA of identical size in ACTH-secreting bronchial carcinoids and pituitary tumors. Other types of nonpituitary tumors responsible for ectopic ACTH syndrome presented much lower levels of both POMC and V3 receptor gene expression than those found in ACTH-secreting bronchial carcinoids. In contrast with the V3 receptor, CRH receptor mRNA was detected in the majority of neuroendocrine tumors irrespective of their POMC status. These results show that expression of the V3 receptor gene participates in the corticotroph phenotype. Its striking association with ACTH-secreting bronchial carcinoids defines a subset of nonpituitary tumors in which ectopic POMC gene expression is but one aspect of a wider process of corticotroph cell differentiation, and opens new possibilities of pharmacological investigations and even manipulations of this peculiar ACTH hypersecretory syndrome. PMID:8636444

The pituitary V3 vasopressin receptor and the corticotroph phenotype in ectopic ACTH syndrome.

PubMed

de Keyzer, Y; Lenne, F; Auzan, C; Jégou, S; René, P; Vaudry, H; Kuhn, J M; Luton, J P; Clauser, E; Bertagna, X

1996-03-01

Ectopic ACTH secretion occurs in highly differentiated and rather indolent tumors like bronchial carcinoids or, in contrast, in various types of aggressive and poorly differentiated neuroendocrine tumors. We explored this phenomenon using the recently cloned human pituitary V3 vasopressin receptor as an alternate molecular marker of the corticotroph phenotype. Expression of V3 receptor, corticotrophin releasing hormone (CRH) receptor, and proopiomelanocortin (POMC) genes was examined in tumors of pituitary and nonpituitary origin. A comparative RT-PCR approach revealed signals for both V3 receptor and CHR receptor mRNAs in 17 of 18 ACTH-secreting pituitary adenomas, and 6 of 6 normal pituitaries; in six growth hormone- or prolactin-secreting adenomas, a very faint V3 receptor signal was observed in three cases, and CRH receptor signal was undetected in all. Six of eight bronchial carcinoids responsible for the ectopic ACTH syndrome had both POMC and V3 receptor signals as high as those in ACTH-secreting pituitary adenomas; in contrast, no POMC signal and only a very faint V3 receptor signal were detected in six of eight nonsecreting bronchial carcinoids. Northern blot analysis showed V3 receptor mRNA of identical size in ACTH-secreting bronchial carcinoids and pituitary tumors. Other types of nonpituitary tumors responsible for ectopic ACTH syndrome presented much lower levels of both POMC and V3 receptor gene expression than those found in ACTH-secreting bronchial carcinoids. In contrast with the V3 receptor, CRH receptor mRNA was detected in the majority of neuroendocrine tumors irrespective of their POMC status. These results show that expression of the V3 receptor gene participates in the corticotroph phenotype. Its striking association with ACTH-secreting bronchial carcinoids defines a subset of nonpituitary tumors in which ectopic POMC gene expression is but one aspect of a wider process of corticotroph cell differentiation, and opens new possibilities of pharmacological investigations and even manipulations of this peculiar ACTH hypersecretory syndrome.

Ectopic Expression of Homeobox Gene NKX2-1 in Diffuse Large B-Cell Lymphoma Is Mediated by Aberrant Chromatin Modifications

PubMed Central

Nagel, Stefan; Ehrentraut, Stefan; Tomasch, Jürgen; Quentmeier, Hilmar; Meyer, Corinna; Kaufmann, Maren; Drexler, Hans G.; MacLeod, Roderick A. F.

2013-01-01

Homeobox genes encode transcription factors ubiquitously involved in basic developmental processes, deregulation of which promotes cell transformation in multiple cancers including hematopoietic malignancies. In particular, NKL-family homeobox genes TLX1, TLX3 and NKX2-5 are ectopically activated by chromosomal rearrangements in T-cell neoplasias. Here, using transcriptional microarray profiling and RQ-PCR we identified ectopic expression of NKL-family member NKX2-1, in a diffuse large B-cell lymphoma (DLBCL) cell line SU-DHL-5. Moreover, in silico analysis demonstrated NKX2-1 overexpression in 5% of examined DLBCL patient samples. NKX2-1 is physiologically expressed in lung and thyroid tissues where it regulates differentiation. Chromosomal and genomic analyses excluded rearrangements at the NKX2-1 locus in SU-DHL-5, implying alternative activation. Comparative expression profiling implicated several candidate genes in NKX2-1 regulation, variously encoding transcription factors, chromatin modifiers and signaling components. Accordingly, siRNA-mediated knockdown and overexpression studies confirmed involvement of transcription factor HEY1, histone methyltransferase MLL and ubiquitinated histone H2B in NKX2-1 deregulation. Chromosomal aberrations targeting MLL at 11q23 and the histone gene cluster HIST1 at 6p22 which we observed in SU-DHL-5 may, therefore, represent fundamental mutations mediating an aberrant chromatin structure at NKX2-1. Taken together, we identified ectopic expression of NKX2-1 in DLBCL cells, representing the central player in an oncogenic regulative network compromising B-cell differentiation. Thus, our data extend the paradigm of NKL homeobox gene deregulation in lymphoid malignancies. PMID:23637834

Subcellular targeting of p33ING1b by phosphorylation-dependent 14-3-3 binding regulates p21WAF1 expression.

PubMed

Gong, Wei; Russell, Michael; Suzuki, Keiko; Riabowol, Karl

2006-04-01

ING1 is a type II tumor suppressor that affects cell growth, stress signaling, apoptosis, and DNA repair by altering chromatin structure and regulating transcription. Decreased ING1 expression is seen in several human cancers, and mislocalization has been noted in diverse types of cancer cells. Aberrant targeting may, therefore, functionally inactivate ING1. Bioinformatics analysis identified a sequence between the nuclear localization sequence and plant homeodomain domains of ING1 that closely matched the binding motif of 14-3-3 proteins that target cargo proteins to specific subcellular locales. We find that the widely expressed p33(ING1b) splicing isoform of ING1 interacts with members of the 14-3-3 family of proteins and that this interaction is regulated by the phosphorylation status of ING1. 14-3-3 binding resulted in significant amounts of p33(ING1b) protein being tethered in the cytoplasm. As shown previously, ectopic expression of p33(ING1b) increased levels of the p21(Waf1) cyclin-dependent kinase inhibitor upon UV-induced DNA damage. Overexpression of 14-3-3 inhibited the up-regulation of p21(Waf1) by p33(ING1b), consistent with the idea that mislocalization blocks at least one of ING1's biological activities. These data support the idea that the 14-3-3 proteins play a crucial role in regulating the activity of p33(ING1b) by directing its subcellular localization.

Functional and Evolutionary Analysis of the CASPARIAN STRIP MEMBRANE DOMAIN PROTEIN Family1[C][W

PubMed Central

Roppolo, Daniele; Boeckmann, Brigitte; Pfister, Alexandre; Boutet, Emmanuel; Rubio, Maria C.; Dénervaud-Tendon, Valérie; Vermeer, Joop E.M.; Gheyselinck, Jacqueline; Xenarios, Ioannis; Geldner, Niko

2014-01-01

CASPARIAN STRIP MEMBRANE DOMAIN PROTEINS (CASPs) are four-membrane-span proteins that mediate the deposition of Casparian strips in the endodermis by recruiting the lignin polymerization machinery. CASPs show high stability in their membrane domain, which presents all the hallmarks of a membrane scaffold. Here, we characterized the large family of CASP-like (CASPL) proteins. CASPLs were found in all major divisions of land plants as well as in green algae; homologs outside of the plant kingdom were identified as members of the MARVEL protein family. When ectopically expressed in the endodermis, most CASPLs were able to integrate the CASP membrane domain, which suggests that CASPLs share with CASPs the propensity to form transmembrane scaffolds. Extracellular loops are not necessary for generating the scaffold, since CASP1 was still able to localize correctly when either one of the extracellular loops was deleted. The CASP first extracellular loop was found conserved in euphyllophytes but absent in plants lacking Casparian strips, an observation that may contribute to the study of Casparian strip and root evolution. In Arabidopsis (Arabidopsis thaliana), CASPL showed specific expression in a variety of cell types, such as trichomes, abscission zone cells, peripheral root cap cells, and xylem pole pericycle cells. PMID:24920445

Functional and Evolutionary Analysis of the CASPARIAN STRIP MEMBRANE DOMAIN PROTEIN Family.

PubMed

Roppolo, Daniele; Boeckmann, Brigitte; Pfister, Alexandre; Boutet, Emmanuel; Rubio, Maria C; Dénervaud-Tendon, Valérie; Vermeer, Joop E M; Gheyselinck, Jacqueline; Xenarios, Ioannis; Geldner, Niko

2014-08-01

CASPARIAN STRIP MEMBRANE DOMAIN PROTEINS (CASPs) are four-membrane-span proteins that mediate the deposition of Casparian strips in the endodermis by recruiting the lignin polymerization machinery. CASPs show high stability in their membrane domain, which presents all the hallmarks of a membrane scaffold. Here, we characterized the large family of CASP-like (CASPL) proteins. CASPLs were found in all major divisions of land plants as well as in green algae; homologs outside of the plant kingdom were identified as members of the MARVEL protein family. When ectopically expressed in the endodermis, most CASPLs were able to integrate the CASP membrane domain, which suggests that CASPLs share with CASPs the propensity to form transmembrane scaffolds. Extracellular loops are not necessary for generating the scaffold, since CASP1 was still able to localize correctly when either one of the extracellular loops was deleted. The CASP first extracellular loop was found conserved in euphyllophytes but absent in plants lacking Casparian strips, an observation that may contribute to the study of Casparian strip and root evolution. In Arabidopsis (Arabidopsis thaliana), CASPL showed specific expression in a variety of cell types, such as trichomes, abscission zone cells, peripheral root cap cells, and xylem pole pericycle cells. © 2014 American Society of Plant Biologists. All Rights Reserved.

CIKS, a connection to Ikappa B kinase and stress-activated protein kinase.

PubMed

Leonardi, A; Chariot, A; Claudio, E; Cunningham, K; Siebenlist, U

2000-09-12

Pathogens, inflammatory signals, and stress cause acute transcriptional responses in cells. The induced expression of genes in response to these signals invariably involves transcription factors of the NF-kappaB and AP-1/ATF families. Activation of NF-kappaB factors is thought to be mediated primarily via IkappaB kinases (IKK), whereas that of AP-1/ATF can be mediated by stress-activated protein kinases (SAPKs; also named Jun kinases or JNKs). IKKalpha and IKKbeta are two catalytic subunits of a core IKK complex that also contains the regulatory subunit NEMO (NF-kappaB essential modulator)/IKKgamma. The latter protein is essential for activation of the IKKs, but its mechanism of action is not known. Here we describe the molecular cloning of CIKS (connection to IKK and SAPK/JNK), a previously unknown protein that directly interacts with NEMO/IKKgamma in cells. When ectopically expressed, CIKS stimulates IKK and SAPK/JNK kinases and it transactivates an NF-kappaB-dependent reporter. Activation of NF-kappaB is prevented in the presence of kinase-deficient, interfering mutants of the IKKs. CIKS may help to connect upstream signaling events to IKK and SAPK/JNK modules. CIKS could coordinate the activation of two stress-induced signaling pathways, functions reminiscent of those noted for tumor necrosis factor receptor-associated factor adaptor proteins.

Expression of the cancer-testis antigen BORIS correlates with prostate cancer.

PubMed

Cheema, Zubair; Hari-Gupta, Yukti; Kita, Georgia-Xanthi; Farrar, Dawn; Seddon, Ian; Corr, John; Klenova, Elena

2014-02-01

BORIS, a paralogue of the transcription factor CTCF, is a member of the cancer-testis antigen (CT) family. BORIS is normally present at high levels in the testis; however it is aberrantly expressed in various tumors and cancer cell lines. The main objectives of this study were to investigate BORIS expression together with sub-cellular localization in both prostate cell lines and tumor tissues, and assess correlations between BORIS and clinical/pathological characteristics. We examined BORIS mRNA expression, protein levels and cellular localization in a panel of human prostate tissues, cancer and benign, together with a panel prostate cell lines. We also compared BORIS levels and localization with clinical/pathological characteristics in prostate tumors. BORIS was detected in all inspected prostate cancer cell lines and tumors, but was absent in benign prostatic hyperplasia. Increased levels of BORIS protein positively correlated with Gleason score, T-stage and androgen receptor (AR) protein levels in prostate tumors. The relationship between BORIS and AR was further highlighted in prostate cell lines by the ability of ectopically expressed BORIS to activate the endogenous AR mRNA and protein. BORIS localization in the nucleus plus cytoplasm was also associated with higher BORIS levels and Gleason score. Detection of BORIS in prostate tumors suggests potential applications of BORIS as a biomarker for prostate cancer diagnosis, as an immunotherapy target and, potentially, a prognostic marker of more aggressive prostate cancer. The ability of BORIS to activate the AR gene indicates BORIS involvement in the growth and development of prostate tumors. © 2013 Wiley Periodicals, Inc.

IL-7Rα and E47: independent pathways required for development of multipotent lymphoid progenitors

PubMed Central

Kee, Barbara L.; Bain, Gretchen; Murre, Cornelis

2002-01-01

Mice that lack the transcription factors encoded by the E2A gene or the receptor for interleukin 7 (IL-7R) have severe overlapping defects in lymphocyte development. Here, we show that E2A proteins are required for the survival of early T-lineage cells; however, they function through a pathway that is distinct from the survival pathway initiated by IL-7R signaling. While E2A proteins are required to suppress caspase 3 activation, ectopic expression of the anti-apoptotic protein Bcl-2 is not sufficient to overcome the lymphopoietic defects observed in the absence of E2A. Remarkably, mice that lack both IL-7Rα and E47 display a synergistic decrease in the number of T-cell, NK-cell and multipotent progenitors in the thymus, indicating that these distinct survival pathways converge to promote the development of multipotent lymphoid progenitors. PMID:11782430

Positional cloning identifies zebrafish one-eyed pinhead as a permissive EGF-related ligand required during gastrulation.

PubMed

Zhang, J; Talbot, W S; Schier, A F

1998-01-23

The zebrafish one-eyed pinhead (oep) mutation disrupts embryonic development, resulting in cyclopia and defects in endoderm, prechordal plate, and ventral neuroectoderm formation. We report the molecular isolation of oep using a positional cloning approach. The oep gene encodes a novel EGF-related protein with similarity to the EGF-CFC proteins cripto, cryptic, and FRL-1. Wild-type oep protein contains a functional signal sequence and is membrane-associated. Following ubiquitous maternal and zygotic expression, highest levels of oep mRNA are found in the gastrula margin and in axial structures and forebrain. Widespread misexpression of both membrane-attached and secreted forms of oep rescues prechordal plate and forebrain development in mutant embryos but does not lead to the ectopic induction of these cell types in wild-type fish. These results establish an essential but permissive role for an EGF-related ligand during vertebrate gastrulation.

The Drosophila Midkine/Pleiotrophin Homologues Miple1 and Miple2 Affect Adult Lifespan but Are Dispensable for Alk Signaling during Embryonic Gut Formation

PubMed Central

Hugosson, Fredrik; Sjögren, Camilla; Birve, Anna; Hedlund, Ludmilla; Eriksson, Therese; Palmer, Ruth H.

2014-01-01

Midkine (MDK) and Pleiotrophin (PTN) are small heparin-binding cytokines with closely related structures. The Drosophila genome harbours two genes encoding members of the MDK/PTN family of proteins, known as miple1 and miple2. We have investigated the role of Miple proteins in vivo, in particular with regard to their proposed role as ligands for the Alk receptor tyrosine kinase (RTK). Here we show that Miple proteins are neither required to drive Alk signaling during Drosophila embryogenesis, nor are they essential for development in the fruit fly. Additionally we show that neither MDK nor PTN can activate hALK in vivo when ectopically co-expressed in the fly. In conclusion, our data suggest that Alk is not activated by MDK/PTN related growth factors Miple1 and Miple 2 in vivo. PMID:25380037

The Drosophila midkine/pleiotrophin homologues Miple1 and Miple2 affect adult lifespan but are dispensable for alk signaling during embryonic gut formation.

PubMed

Hugosson, Fredrik; Sjögren, Camilla; Birve, Anna; Hedlund, Ludmilla; Eriksson, Therese; Palmer, Ruth H

2014-01-01

Midkine (MDK) and Pleiotrophin (PTN) are small heparin-binding cytokines with closely related structures. The Drosophila genome harbours two genes encoding members of the MDK/PTN family of proteins, known as miple1 and miple2. We have investigated the role of Miple proteins in vivo, in particular with regard to their proposed role as ligands for the Alk receptor tyrosine kinase (RTK). Here we show that Miple proteins are neither required to drive Alk signaling during Drosophila embryogenesis, nor are they essential for development in the fruit fly. Additionally we show that neither MDK nor PTN can activate hALK in vivo when ectopically co-expressed in the fly. In conclusion, our data suggest that Alk is not activated by MDK/PTN related growth factors Miple1 and Miple 2 in vivo.

Identification of p90 Ribosomal S6 Kinase 2 as a Novel Host Protein in HBx Augmenting HBV Replication by iTRAQ-Based Quantitative Comparative Proteomics.

PubMed

Yan, Li-Bo; Yu, You-Jia; Zhang, Qing-Bo; Tang, Xiao-Qiong; Bai, Lang; Huang, FeiJun; Tang, Hong

2018-05-01

The aim of this study was to screen for novel host proteins that play a role in HBx augmenting Hepatitis B virus (HBV) replication. Three HepG2 cell lines stably harboring different functional domains of HBx (HBx, HBx-Cm6, and HBx-Cm16) were cultured. ITRAQ technology integrated with LC-MS/MS analysis was applied to identify the proteome differences among these three cell lines. In brief, a total of 70 different proteins were identified among HepG2-HBx, HepG2-HBx-Cm6, and HepG2-HBx-Cm16 by double repetition. Several differentially expressed proteins, including p90 ribosomal S6 kinase 2 (RSK2), were further validated. RSK2 was expressed at higher levels in HepG2-HBx and HepG2-HBx-Cm6 compared with HepG2-HBx-Cm16. Furthermore, levels of HBV replication intermediates were decreased after silencing RSK2 in HepG2.2.15. An HBx-minus HBV mutant genome led to decreased levels of HBV replication intermediates and these decreases were restored to levels similar to wild-type HBV by transient ectopic expression of HBx. After silencing RSK2 expression, the levels of HBV replication intermediates synthesized from the HBx-minus HBV mutant genome were not restored to levels that were observed with wild-type HBV by transient HBx expression. Based on iTRAQ quantitative comparative proteomics, RSK2 was identified as a novel host protein that plays a role in HBx augmenting HBV replication. © 2018 The Authors. Proteomics - Clinical Application Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A Herpesvirus Ribosome-Associated, RNA-Binding Protein Confers a Growth Advantage upon Mutants Deficient in a GADD34-Related Function†

PubMed Central

Mulvey, Matthew; Poppers, Jeremy; Ladd, Alison; Mohr, Ian

1999-01-01

The herpes simplex virus type 1 γ34.5 gene product and the cellular GADD34 protein both contain similar domains that can regulate the activity of eukaryotic initiation factor 2 (eIF2), a critical translation initiation factor. Viral mutants that lack the GADD34-related function grow poorly on a variety of malignant human cells, as activation of the cellular PKR kinase leads to the accumulation of inactive, phosphorylated eIF2 at late times postinfection. Termination of translation prior to the completion of the viral reproductive cycle leads to impaired growth. Extragenic suppressors that regain the ability to synthesize proteins efficiently in the absence of the viral GADD34-related function have been isolated. These suppressor alleles are dominant in trans and affect the steady-state accumulation of several viral mRNA species. We demonstrate that deregulated expression of Us11, a virus-encoded RNA-binding, ribosome-associated protein is necessary and sufficient to confer a growth advantage upon viral mutants that lack a GADD34-related function. Ectopic expression of Us11 reduces the accumulation of the activated cellular PKR kinase and allows for sustained protein synthesis. Thus, an RNA-binding, ribosome-associated protein (Us11) and a GADD34-related protein (γ34.5) both function in a signal pathway that regulates translation by modulating eIF2 phosphorylation. PMID:10074192

Immortalization of pig fibroblast cells by transposon-mediated ectopic expression of porcine telomerase reverse transcriptase.

PubMed

He, Shan; Li, Yangyang; Chen, Yang; Zhu, Yue; Zhang, Xinyu; Xia, Xiaoli; Sun, Huaichang

2016-08-01

Pigs are the most economically important livestock, but pig cell lines useful for physiological studies and/or vaccine development are limited. Although several pig cell lines have been generated by oncogene transformation or human telomerase reverse transcriptase (TERT) immortalization, these cell lines contain viral sequences and/or antibiotic resistance genes. In this study, we established a new method for generating pig cell lines using the Sleeping Beauty (SB) transposon-mediated ectopic expression of porcine telomerase reverse transcriptase (pTERT). The performance of the new method was confirmed by generating a pig fibroblast cell (PFC) line. After transfection of primary PFCs with the SB transposon system, one cell clone containing the pTERT expression cassette was selected by dilution cloning and passed for different generations. After passage for more than 40 generations, the cell line retained stable expression of ectopic pTERT and continuous growth potential. Further characterization showed that the cell line kept the fibroblast morphology, growth curve, population doubling time, cloning efficiency, marker gene expression pattern, cell cycle distribution and anchorage-dependent growth property of the primary cells. These data suggest that the new method established is useful for generating pig cell lines without viral sequence and antibiotic resistant gene.

Hepatic leukemia factor promotes resistance to cell death: Implications for therapeutics and chronotherapy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Waters, Katrina M.; Sontag, Ryan L.; Weber, Thomas J., E-mail: Thomas.Weber@pnl.gov

Physiological variation related to circadian rhythms and aberrant gene expression patterns are believed to modulate therapeutic efficacy, but the precise molecular determinants remain unclear. Here we examine the regulation of cell death by hepatic leukemia factor (HLF), which is an output regulator of circadian rhythms and is aberrantly expressed in human cancers, using an ectopic expression strategy in JB6 mouse epidermal cells and human keratinocytes. Ectopic HLF expression inhibited cell death in both JB6 cells and human keratinocytes, as induced by serum-starvation, tumor necrosis factor alpha and ionizing radiation. Microarray analysis indicates that HLF regulates a complex multi-gene transcriptional programmore » encompassing upregulation of anti-apoptotic genes, downregulation of pro-apoptotic genes, and many additional changes that are consistent with an anti-death program. Collectively, our results demonstrate that ectopic expression of HLF, an established transcription factor that cycles with circadian rhythms, can recapitulate many features associated with circadian-dependent physiological variation. - Highlights: ► Circadian-dependent physiological variation impacts therapeutic efficacy. ► Hepatic leukemia factor inhibits cell death and is a candidate circadian factor. ► Hepatic leukemia factor anti-death program is conserved in murine and human cells. ► Transcriptomics indicates the anti-death program results from a systems response.« less

Porcine deltacoronavirus accessory protein NS6 antagonizes IFN-β production by interfering with the binding of RIG-I/MDA5 to double-stranded RNA.

PubMed

Fang, Puxian; Fang, Liurong; Ren, Jie; Hong, Yingying; Liu, Xiaorong; Zhao, Yunyang; Wang, Dang; Peng, Guiqing; Xiao, Shaobo

2018-05-16

Porcine deltacoronavirus (PDCoV) has recently emerged as an enteric pathogen that can cause serious vomiting and diarrhea in suckling piglets. The first outbreak of PDCoV occurred in the United States in 2014 and was followed by reports of PDCoV in South Korea, China, Thailand, Lao people's Democratic Republic, and Vietnam, leading to economic losses for pig farms and posing considerable threat to the swine industry worldwide. Our previous studies have shown that PDCoV encodes three accessory proteins, NS6, NS7, and NS7a, but the functions of these proteins in viral replication, pathogenesis, and immune regulation remain unclear. Here, we found that ectopic expression of accessory protein NS6 significantly inhibits Sendai virus-induced interferon-β (IFN-β) production, as well as the activation of transcription factors IRF3 and NF-κB. Interestingly, NS6 does not impede the IFN-β promoter activation mediated via key molecules in the RIG-I-like receptor (RLR) signaling pathway, specifically RIG-I, MDA5, and their downstream molecules MAVS, TBK1, IKKϵ, and IRF3. Further analyses revealed that NS6 is not a RNA-binding protein; however, it interacts with RIG-I/MDA5. This interaction attenuates the binding of double-stranded RNA by RIG-I/MDA5, resulting in the reduction of RLR-mediated IFN-β production. Taken together, our results demonstrate that ectopic expression of NS6 antagonizes IFN-β production by interfering with the binding of RIG-I/MDA5 to double-stranded RNA, revealing a new strategy employed by PDCoV accessory proteins to counteract the host innate antiviral immune response. IMPORTANCE Coronavirus accessory proteins are species-specific, and they perform multiple functions in viral pathogenicity and immunity, such as acting as interferon (IFN) antagonists and cell death inducers. Our previous studies have shown that porcine deltacoronavirus (PDCoV) encodes three accessory proteins. Here, we demonstrated for the first time that PDCoV accessory protein NS6 antagonizes IFN-β production by interacting with RIG-I and MDA5 to impede their association with double-stranded RNA. This is an efficient strategy of antagonizing type I IFN production by disrupting the binding of host pattern recognition receptors (PRRs) and pathogen-associated molecular patterns (PAMPs). These findings deepen our understanding of the function of accessory protein NS6 and may direct us toward novel therapeutic targets and lead to the development of more effective vaccines against PDCoV infection. Copyright © 2018 American Society for Microbiology.

Methylation of PLCD1 and adenovirus-mediated PLCD1 overexpression elicits a gene therapy effect on human breast cancer

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mu, Haixi; Department of Endocrine and breast Surgery, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016; Wang, Na

Our previous study showed that PLCD1 significantly decreases cell proliferation and affects cell cycle progression in breast cancer cells. In the present study, we aimed to investigate its functional and molecular mechanisms, and whether or not can become a new target for gene therapies. We found reduced PLCD1 protein expression in breast tumor tissues compared with paired surgical margin tissues. PLCD1 promoter CpG methylation was detected in 55 of 96 (57%) primary breast tumors, but not in surgical-margin tissues and normal breast tissues. Ectopic expression of PLCD1 inhibited breast tumor cell proliferation in vivo by inducing apoptosis and suppressed tumormore » cell migration by regulating cytoskeletal reorganization proteins including RhoA and phospho-cofilin. Furthermore, we found that PLCD1 induced p53 accumulation, increased p27 and p21 protein levels, and cleaved PARP. Finally, we constructed an adenoviral vector expressing PLCD1 (AdH5-PLCD1), which exhibited strong cytotoxicity in breast cancer cells. Our findings provide insights into the development of PLCD1 gene therapies for breast cancer and perhaps, other human cancers. - Highlights: • PLCD1 is downregulated via hypermethylation in breast cancer. • PLCD1 suppressed cell migration by regulating cytoskeletal reorganization proteins. • Adenovirus AdHu5-PLCD1 may be a novel therapeutic option for breast cancer.« less

The Polerovirus F box protein P0 targets ARGONAUTE1 to suppress RNA silencing.

PubMed

Bortolamiol, Diane; Pazhouhandeh, Maghsoud; Marrocco, Katia; Genschik, Pascal; Ziegler-Graff, Véronique

2007-09-18

Plants employ post-transcriptional gene silencing (PTGS) as an antiviral defense response. In this mechanism, viral-derived small RNAs are incorporated into the RNA-induced silencing complex (RISC) to guide degradation of the corresponding viral RNAs. ARGONAUTE1 (AGO1) is a key component of RISC: it carries the RNA slicer activity. As a counter-defense, viruses have evolved various proteins that suppress PTGS. Recently, we showed that the Polerovirus P0 protein carries an F box motif required to form an SCF-like complex, which is also essential for P0's silencing suppressor function. Here, we investigate the molecular mechanism by which P0 impairs PTGS. First we show that P0's expression does not affect the biogenesis of primary siRNAs in an inverted repeat-PTGS assay, but it does affect their activity. Moreover, P0's expression in transformed Arabidopsis plants leads to various developmental abnormalities reminiscent of mutants affected in miRNA pathways, which is accompanied by enhanced levels of several miRNA-target transcripts, suggesting that P0 acts at the level of RISC. Interestingly, ectopic expression of P0 triggered AGO1 protein decay in planta. Finally, we provide evidence that P0 physically interacts with AGO1. Based on these results, we propose that P0 hijacks the host SCF machinery to modulate gene silencing by destabilizing AGO1.

SMILE upregulated by metformin inhibits the function of androgen receptor in prostate cancer cells.

PubMed

Lee, Seung-Yon; Song, Chin-Hee; Xie, Yuan-Bin; Jung, Chaeyong; Choi, Hueng-Sik; Lee, Keesook

2014-11-28

Metformin, a diabetes drug, has been reported to inhibit the growth of prostate cancer cells. In this study, we investigated the effect and action mechanism of metformin on the function of androgen receptor (AR), a key molecule in the proliferation of prostate cancer cells. Metformin was found to reduce androgen-dependent cell growth and the expression of AR target genes by inhibiting AR function in prostate cancer cells such as LNCaP and C4-2 cells. Interestingly, metformin upregulated the protein level of small heterodimer partner-interacting leucine zipper (SMILE), a coregulator of nuclear receptors, and knockdown of SMILE expression with shRNA abolished the inhibitory effect of metformin on AR function. Further studies revealed that SMILE protein itself suppressed the transactivation of AR, and its ectopic expression resulted in the repressed expression of endogenous AR target genes, PSA and NKX3.1, in LNCaP cells. In addition, SMILE protein physically interacted with AR and competed with the AR coactivator SRC-1 to modulate AR transactivation. As expected, SMILE repressed androgen-dependent growth of LNCaP and C4-2 cells. Taken together, these results suggest that SMILE, which is induced by metformin, functions as a novel AR corepressor and may mediate the inhibitory effect of metformin on androgen-dependent growth of prostate cancer cells. Copyright © 2014. Published by Elsevier Ireland Ltd.

Drosophila gene tao-1 encodes proteins with and without a Ste20 kinase domain that affect cytoskeletal architecture and cell migration differently

PubMed Central

Pflanz, Ralf; Voigt, Aaron; Yakulov, Toma; Jäckle, Herbert

2015-01-01

Tao-1, the single representative of the Sterile 20 kinase subfamily in Drosophila, is best known for destabilizing microtubules at the actin-rich cortex, regulating the cytoskeletal architecture of cells. More recently, Tao-1 was shown to act in the Salvador–Warts–Hippo pathway by phosphorylating Hippo, regulating cell growth as well as cell polarity. Here, we show that tao-1 encodes two proteins, one with the Sterile 20 kinase domain (Tao-L) and one without it (Tao-S), and that they act in an antagonistic manner. Tao-L expression causes lamellipodia-like cell protrusions, whereas Tao-S expression results in filopodia-like structures that make cells stick to the surface they attach to. Ectopic Tao-1 expression in the anterior region of Drosophila embryos results in pole cell formation as normally observed at the posterior end. Tao-S expression causes primordial germ cells (PGCs) to adhere to the inner wall of the gut primordia and prevents proper transepithelial migration to the gonads. Conversely, RNAi knockdowns of Tao-1 cause disordered migration of PGCs out of the gut epithelium, their dispersal within the embryo and cell death. The results reveal a novel function of Tao-1 in cell migration, which is based on antagonistic activities of two proteins encoded by a single gene. PMID:25589578

Role of an expansin-like molecule in Dictyostelium morphogenesis and regulation of its gene expression by the signal transducer and activator of transcription protein Dd-STATa.

PubMed

Ogasawara, Shun; Shimada, Nao; Kawata, Takefumi

2009-02-01

Expansins are proteins involved in plant morphogenesis, exerting their effects on cellulose to extend cell walls. Dictyostelium is an organism that possesses expansin-like molecules, but their functions are not known. In this study, we analyzed the expL7 (expansin-like 7) gene, which has been identified as a putative target of Dd-STATa, a Dictyostelium homolog of the metazoan signal transducer and activator of transcription (STAT) proteins. Promoter fragments of the expL7 were fused to a lacZ reporter and the expression patterns determined. As expected from the behavior of the endogenous expL7 gene, the expL7/lacZ fusion gene was downregulated in Dd-STATa null slugs. In the parental strain, the expL7 promoter was activated in the anterior tip region. Mutational analysis of the promoter identified a sequence that was necessary for expression in tip cells. In addition, an activator sequence for pstAB cells was identified. These sequences act in combination with the repressor region to prevent ectopic expL7 expression in the prespore and prestalk regions of the slug and culminant. Although the expL7 null mutant showed no phenotypic change, the expL7 overexpressor showed aberrant stalk formation. These results indicate that the expansin-like molecule is important for morphogenesis in Dictyostelium.

The TRANSPARENT TESTA16 Locus Encodes the ARABIDOPSIS BSISTER MADS Domain Protein and Is Required for Proper Development and Pigmentation of the Seed Coat

PubMed Central

Nesi, Nathalie; Debeaujon, Isabelle; Jond, Clarisse; Stewart, Amanda J.; Jenkins, Gareth I.; Caboche, Michel; Lepiniec, Loïc

2002-01-01

Screening for seed pigmentation phenotypes in Arabidopsis led to the isolation of three allelic yellow-seeded mutants, which defined the novel TRANSPARENT TESTA16 (TT16) locus. Cloning of TT16 was performed by T-DNA tagging and confirmed by genetic complementation and sequencing of two mutant alleles. TT16 encodes the ARABIDOPSIS BSISTER (ABS) MADS domain protein. ABS belongs to the recently identified “B-sister” (BS) clade, which contains genes of unknown function that are expressed mainly in female organs. Phylogenetic analyses using a maximum parsimony approach confirmed that TT16/ABS and related proteins form a monophyletic group. TT16/ABS was expressed mainly in the ovule, as are the other members of the BS clade. TT16/ABS is necessary for BANYULS expression and proanthocyanidin accumulation in the endothelium of the seed coat, with the exception of the chalazal-micropylar area. In addition, mutant phenotype and ectopic expression analyses suggested that TT16/ABS also is involved in the specification of endothelial cells. Nevertheless, TT16/ABS apparently is not required for proper ovule function. We report the functional characterization of a member of the BS MADS box gene subfamily, demonstrating its involvement in endothelial cell specification as well as in the increasingly complex genetic control of flavonoid biosynthesis in the Arabidopsis seed coat. PMID:12368498

N-acetylcysteine negatively regulates Notch3 and its malignant signaling

PubMed Central

Zhu, Juan-Juan; Liu, Xue-Xia; You, Hui; Gong, Mei-Ying; Zou, Ming; Cheng, Wen-Hsing; Zhu, Jian-Hong

2016-01-01

Notch3 receptor is expressed in a variety of cancers and the excised active intracellular domain (N3ICD) initiates its signaling cascade. N-acetylcysteine (NAC) as an antioxidant has been implicated in cancer prevention and therapy. In this study, we demonstrated a negative regulation of Notch3 by NAC in cancer cells. HeLa cells treated with NAC exhibited a time- and concentration-dependent decrease in Notch3 levels and its downstream effectors Hes1 and HRT1 in a manner independent of f-secretase or glutathione. In contrast, NAC did not affect protein levels of Notch1, the full length Notch3 precursor, or ectopically expressed N3ICD. Although SOD, catalase and NAC suppressed reactive oxygen species in HeLa cells, the first two antioxidants did not impact on Notch3 levels. While the mRNA expression of Notch3 was not altered by NAC, functional inhibition of lysosome, but not proteasome, blocked the NAC-dependent reduction of Notch3 levels. Furthermore, results from Notch3 silencing and N3ICD overexpression demonstrated that NAC prevented malignant phenotypes through down-regulation of Notch3 protein in multiple cancer cells. In summary, NAC reduces Notch3 levels through lysosome-dependent protein degradation, thereby negatively regulates Notch3 malignant signaling in cancer cells. These results implicate a novel NAC treatment in sensitizing Notch3-expressing tumors. PMID:27102435

N-acetylcysteine negatively regulates Notch3 and its malignant signaling.

PubMed

Zhang, Xiong; Wang, Ya-Nan; Zhu, Juan-Juan; Liu, Xue-Xia; You, Hui; Gong, Mei-Ying; Zou, Ming; Cheng, Wen-Hsing; Zhu, Jian-Hong

2016-05-24

Notch3 receptor is expressed in a variety of cancers and the excised active intracellular domain (N3ICD) initiates its signaling cascade. N-acetylcysteine (NAC) as an antioxidant has been implicated in cancer prevention and therapy. In this study, we demonstrated a negative regulation of Notch3 by NAC in cancer cells. HeLa cells treated with NAC exhibited a time- and concentration-dependent decrease in Notch3 levels and its downstream effectors Hes1 and HRT1 in a manner independent of f-secretase or glutathione. In contrast, NAC did not affect protein levels of Notch1, the full length Notch3 precursor, or ectopically expressed N3ICD. Although SOD, catalase and NAC suppressed reactive oxygen species in HeLa cells, the first two antioxidants did not impact on Notch3 levels. While the mRNA expression of Notch3 was not altered by NAC, functional inhibition of lysosome, but not proteasome, blocked the NAC-dependent reduction of Notch3 levels. Furthermore, results from Notch3 silencing and N3ICD overexpression demonstrated that NAC prevented malignant phenotypes through down-regulation of Notch3 protein in multiple cancer cells. In summary, NAC reduces Notch3 levels through lysosome-dependent protein degradation, thereby negatively regulates Notch3 malignant signaling in cancer cells. These results implicate a novel NAC treatment in sensitizing Notch3-expressing tumors.

T-cell-restricted intracellular antigen 1 facilitates mitochondrial fragmentation by enhancing the expression of mitochondrial fission factor

PubMed Central

Tak, Hyosun; Eun, Jung Woo; Kim, Jihye; Park, So Jung; Kim, Chongtae; Ji, Eunbyul; Lee, Heejin; Kang, Hoin; Cho, Dong-Hyung; Lee, Kyungbun; Kim, Wook; Nam, Suk Woo; Lee, Eun Kyung

2017-01-01

Mitochondrial morphology is dynamically regulated by the formation of small fragmented units or interconnected mitochondrial networks, and this dynamic morphological change is a pivotal process in normal mitochondrial function. In the present study, we identified a novel regulator responsible for the regulation of mitochondrial dynamics. An assay using CHANG liver cells stably expressing mitochondrial-targeted yellow fluorescent protein (mtYFP) and a group of siRNAs revealed that T-cell intracellular antigen protein-1 (TIA-1) affects mitochondrial morphology by enhancing mitochondrial fission. The function of TIA-1 in mitochondrial dynamics was investigated through various biological approaches and expression analysis in human specimen. Downregulation of TIA-1-enhanced mitochondrial elongation, whereas ectopic expression of TIA-1 resulted in mitochondria fragmentation. In addition, TIA-1 increased mitochondrial activity, including the rate of ATP synthesis and oxygen consumption. Further, we identified mitochondrial fission factor (MFF) as a direct target of TIA-1, and showed that TIA-1 promotes mitochondrial fragmentation by enhancing MFF translation. TIA-1 null cells had a decreased level of MFF and less mitochondrial Drp1, a critical factor for mitochondrial fragmentation, thereby enhancing mitochondrial elongation. Taken together, our results indicate that TIA-1 is a novel factor that facilitates mitochondrial dynamics by enhancing MFF expression and contributes to mitochondrial dysfunction. PMID:27612012

Samd7 is a cell type-specific PRC1 component essential for establishing retinal rod photoreceptor identity

PubMed Central

Omori, Yoshihiro; Kubo, Shun; Kon, Tetsuo; Furuhashi, Mayu; Narita, Hirotaka; Kominami, Taro; Ueno, Akiko; Tsutsumi, Ryotaro; Chaya, Taro; Yamamoto, Haruka; Suetake, Isao; Ueno, Shinji; Koseki, Haruhiko; Furukawa, Takahisa

2017-01-01

Precise transcriptional regulation controlled by a transcription factor network is known to be crucial for establishing correct neuronal cell identities and functions in the CNS. In the retina, the expression of various cone and rod photoreceptor cell genes is regulated by multiple transcription factors; however, the role of epigenetic regulation in photoreceptor cell gene expression has been poorly understood. Here, we found that Samd7, a rod-enriched sterile alpha domain (SAM) domain protein, is essential for silencing nonrod gene expression through H3K27me3 regulation in rod photoreceptor cells. Samd7-null mutant mice showed ectopic expression of nonrod genes including S-opsin in rod photoreceptor cells and rod photoreceptor cell dysfunction. Samd7 physically interacts with Polyhomeotic homologs (Phc proteins), components of the Polycomb repressive complex 1 (PRC1), and colocalizes with Phc2 and Ring1B in Polycomb bodies. ChIP assays showed a significant decrease of H3K27me3 in the genes up-regulated in the Samd7-deficient retina, showing that Samd7 deficiency causes the derepression of nonrod gene expression in rod photoreceptor cells. The current study suggests that Samd7 is a cell type-specific PRC1 component epigenetically defining rod photoreceptor cell identity. PMID:28900001

Samd7 is a cell type-specific PRC1 component essential for establishing retinal rod photoreceptor identity.

PubMed

Omori, Yoshihiro; Kubo, Shun; Kon, Tetsuo; Furuhashi, Mayu; Narita, Hirotaka; Kominami, Taro; Ueno, Akiko; Tsutsumi, Ryotaro; Chaya, Taro; Yamamoto, Haruka; Suetake, Isao; Ueno, Shinji; Koseki, Haruhiko; Nakagawa, Atsushi; Furukawa, Takahisa

2017-09-26

Precise transcriptional regulation controlled by a transcription factor network is known to be crucial for establishing correct neuronal cell identities and functions in the CNS. In the retina, the expression of various cone and rod photoreceptor cell genes is regulated by multiple transcription factors; however, the role of epigenetic regulation in photoreceptor cell gene expression has been poorly understood. Here, we found that Samd7, a rod-enriched sterile alpha domain (SAM) domain protein, is essential for silencing nonrod gene expression through H3K27me3 regulation in rod photoreceptor cells. Samd7- null mutant mice showed ectopic expression of nonrod genes including S-opsin in rod photoreceptor cells and rod photoreceptor cell dysfunction. Samd7 physically interacts with Polyhomeotic homologs (Phc proteins), components of the Polycomb repressive complex 1 (PRC1), and colocalizes with Phc2 and Ring1B in Polycomb bodies. ChIP assays showed a significant decrease of H3K27me3 in the genes up-regulated in the Samd7 -deficient retina, showing that Samd7 deficiency causes the derepression of nonrod gene expression in rod photoreceptor cells. The current study suggests that Samd7 is a cell type-specific PRC1 component epigenetically defining rod photoreceptor cell identity.

Ehrlichia secretes Etf-1 to induce autophagy and capture nutrients for its growth through RAB5 and class III phosphatidylinositol 3-kinase

PubMed Central

Lin, Mingqun; Liu, Hongyan; Xiong, Qingming; Niu, Hua; Cheng, Zhihui; Yamamoto, Akitsugu; Rikihisa, Yasuko

2016-01-01

Ehrlichia chaffeensis is an obligatory intracellular bacterium that causes a potentially fatal emerging zoonosis, human monocytic ehrlichiosis. E. chaffeensis has a limited capacity for biosynthesis and metabolism and thus depends mostly on host-synthesized nutrients for growth. Although the host cell cytoplasm is rich with these nutrients, as E. chaffeensis is confined within the early endosome-like membrane-bound compartment, only host nutrients that enter the compartment can be used by this bacterium. How this occurs is unknown. We found that ehrlichial replication depended on autophagy induction involving class III phosphatidylinositol 3-kinase (PtdIns3K) activity, BECN1 (Beclin 1), and ATG5 (autophagy-related 5). Ehrlichia acquired host cell preincorporated amino acids in a class III PtdIns3K-dependent manner and ehrlichial growth was enhanced by treatment with rapamycin, an autophagy inducer. Moreover, ATG5 and RAB5A/B/C were routed to ehrlichial inclusions. RAB5A/B/C siRNA knockdown, or overexpression of a RAB5-specific GTPase-activating protein or dominant-negative RAB5A inhibited ehrlichial infection, indicating the critical role of GTP-bound RAB5 during infection. Both native and ectopically expressed ehrlichial type IV secretion effector protein, Etf-1, bound RAB5 and the autophagy-initiating class III PtdIns3K complex, PIK3C3/VPS34, and BECN1, and homed to ehrlichial inclusions. Ectopically expressed Etf-1 activated class III PtdIns3K as in E. chaffeensis infection and induced autophagosome formation, cleared an aggregation-prone mutant huntingtin protein in a class III PtdIns3K-dependent manner, and enhanced ehrlichial proliferation. These data support the notion that E. chaffeensis secretes Etf-1 to induce autophagy to repurpose the host cytoplasm and capture nutrients for its growth through RAB5 and class III PtdIns3K, while avoiding autolysosomal killing. PMID:27541856

Ehrlichia secretes Etf-1 to induce autophagy and capture nutrients for its growth through RAB5 and class III phosphatidylinositol 3-kinase.

PubMed

Lin, Mingqun; Liu, Hongyan; Xiong, Qingming; Niu, Hua; Cheng, Zhihui; Yamamoto, Akitsugu; Rikihisa, Yasuko

2016-11-01

Ehrlichia chaffeensis is an obligatory intracellular bacterium that causes a potentially fatal emerging zoonosis, human monocytic ehrlichiosis. E. chaffeensis has a limited capacity for biosynthesis and metabolism and thus depends mostly on host-synthesized nutrients for growth. Although the host cell cytoplasm is rich with these nutrients, as E. chaffeensis is confined within the early endosome-like membrane-bound compartment, only host nutrients that enter the compartment can be used by this bacterium. How this occurs is unknown. We found that ehrlichial replication depended on autophagy induction involving class III phosphatidylinositol 3-kinase (PtdIns3K) activity, BECN1 (Beclin 1), and ATG5 (autophagy-related 5). Ehrlichia acquired host cell preincorporated amino acids in a class III PtdIns3K-dependent manner and ehrlichial growth was enhanced by treatment with rapamycin, an autophagy inducer. Moreover, ATG5 and RAB5A/B/C were routed to ehrlichial inclusions. RAB5A/B/C siRNA knockdown, or overexpression of a RAB5-specific GTPase-activating protein or dominant-negative RAB5A inhibited ehrlichial infection, indicating the critical role of GTP-bound RAB5 during infection. Both native and ectopically expressed ehrlichial type IV secretion effector protein, Etf-1, bound RAB5 and the autophagy-initiating class III PtdIns3K complex, PIK3C3/VPS34, and BECN1, and homed to ehrlichial inclusions. Ectopically expressed Etf-1 activated class III PtdIns3K as in E. chaffeensis infection and induced autophagosome formation, cleared an aggregation-prone mutant huntingtin protein in a class III PtdIns3K-dependent manner, and enhanced ehrlichial proliferation. These data support the notion that E. chaffeensis secretes Etf-1 to induce autophagy to repurpose the host cytoplasm and capture nutrients for its growth through RAB5 and class III PtdIns3K, while avoiding autolysosomal killing.

Short‐term disuse promotes fatty acid infiltration into skeletal muscle

PubMed Central

Pagano, Allan F.; Brioche, Thomas; Arc‐Chagnaud, Coralie; Demangel, Rémi; Chopard, Angèle

2017-01-01

Abstract Background Many physiological and/or pathological conditions lead to muscle deconditioning, a well‐described phenomenon characterized by a loss of strength and muscle power mainly due to the loss of muscle mass. Fatty infiltrations, or intermuscular adipose tissue (IMAT), are currently well‐recognized components of muscle deconditioning. Despite the fact that IMAT is present in healthy human skeletal muscle, its increase and accumulation are linked to muscle dysfunction. Although IMAT development has been largely attributable to inactivity, the precise mechanisms of its establishment are still poorly understood. Because the sedentary lifestyle that accompanies age‐related sarcopenia may favour IMAT development, deciphering the early processes of muscle disuse is of great importance before implementing strategies to limit IMAT deposition. Methods In our study, we took advantage of the dry immersion (DI) model of severe muscle inactivity to induce rapid muscle deconditioning during a short period. During the DI, healthy adult men (n = 12; age: 32 ± 5) remained strictly immersed, in a supine position, in a controlled thermo‐neutral water bath. Skeletal muscle biopsies were obtained from the vastus lateralis before and after 3 days of DI. Results We showed that DI for only 3 days was able to decrease myofiber cross‐sectional areas (−10.6%). Moreover, protein expression levels of two key markers commonly used to assess IMAT, perilipin, and fatty acid binding protein 4, were upregulated. We also observed an increase in the C/EBPα and PPARγ protein expression levels, indicating an increase in late adipogenic processes leading to IMAT development. While many stem cells in the muscle environment can adopt the capacity to differentiate into adipocytes, fibro‐adipogenic progenitors (FAPs) represent the population that appears to play a major role in IMAT development. In our study, we showed an increase in the protein expression of PDGFRα, the specific cell surface marker of FAPs, in response to 3 days of DI. It is well recognized that an unfavourable muscle environment drives FAPs to ectopic adiposity and/or fibrosis. Conclusions This study is the first to emphasize that during a short period of severe inactivity, muscle deconditioning is associated with IMAT development. Our study also reveals that FAPs could be the main resident muscle stem cell population implicated in ectopic adiposity development in human skeletal muscle. PMID:29248005

BMP signaling is required for development of the ciliary body.

PubMed

Zhao, Shulei; Chen, Qin; Hung, Fang-Cheng; Overbeek, Paul A

2002-10-01

The ciliary body in the eye secretes aqueous humor and glycoproteins of the vitreous body and maintains the intraocular pressure. The ciliary muscle controls the shape of the lens through the ciliary zonules to focus the image onto the retina. During embryonic development, the ciliary epithelium is derived from the optic vesicle, but the molecular signals that control morphogenesis of the ciliary body are unknown. We report that lens-specific expression of a transgenic protein, Noggin, can block BMP signaling in the mouse eye and result in failure in formation of the ciliary processes. Co-expression of transgenic BMP7 restores normal development of the ciliary epithelium. Ectopic expression of Noggin also promotes differentiation of retinal ganglion cells. These results indicate that BMP signaling is required for development of the ciliary body and may also play a role in regulation of neuronal differentiation in the developing eye.

Phloem-specific expression of the lectin gene from Allium sativum confers resistance to the sap-sucker Nilaparvata lugens.

PubMed

Chandrasekhar, Kottakota; Vijayalakshmi, Muvva; Vani, Kalasamudramu; Kaul, Tanushri; Reddy, Malireddy K

2014-05-01

Rice production is severely hampered by insect pests. Garlic lectin gene (ASAL) holds great promise in conferring protection against chewing (lepidopteran) and sap-sucking (homopteran) insect pests. We have developed transgenic rice lines resistant to sap-sucking brown hopper (Nilaparvata lugens) by ectopic expression of ASAL in their phloem tissues. Molecular analyses of T0 lines confirmed stable integration of transgene. T1 lines (NP 1-2, 4-3, 11-6 & 17-7) showed active transcription and translation of ASAL transgene. ELISA revealed ASAL expression was as high as 0.95% of total soluble protein. Insect bioassays on T2 homozygous lines (NP 18 & 32) revealed significant reduction (~74-83%) in survival rate, development and fecundity of brown hoppers in comparison to wild type. Transgenics exhibited enhanced resistance (1-2 score) against brown hoppers, minimal plant damage and no growth penalty or phenotypic abnormalities.

A Collapsin Response Mediator Protein 2 Isoform Controls Myosin II-Mediated Cell Migration and Matrix Assembly by Trapping ROCK II

PubMed Central

Morgan-Fisher, Marie; Wait, Robin; Couchman, John R.; Wewer, Ulla M.

2012-01-01

Collapsin response mediator protein 2 (CRMP-2) is known as a regulator of neuronal polarity and differentiation through microtubule assembly and trafficking. Here, we show that CRMP-2 is ubiquitously expressed and a splice variant (CRMP-2L), which is expressed mainly in epithelial cells among nonneuronal cells, regulates myosin II-mediated cellular functions, including cell migration. While the CRMP-2 short form (CRMP-2S) is recognized as a substrate of the Rho-GTP downstream kinase ROCK in neuronal cells, a CRMP-2 complex containing 2L not only bound the catalytic domain of ROCK II through two binding domains but also trapped and inhibited the kinase. CRMP-2L protein levels profoundly affected haptotactic migration and the actin-myosin cytoskeleton of carcinoma cells as well as nontransformed epithelial cell migration in a ROCK activity-dependent manner. Moreover, the ectopic expression of CRMP-2L but not -2S inhibited fibronectin matrix assembly in fibroblasts. Underlying these responses, CRMP-2L regulated the kinase activity of ROCK II but not ROCK I, independent of GTP-RhoA levels. This study provides a new insight into CRMP-2 as a controller of myosin II-mediated cellular functions through the inhibition of ROCK II in nonneuronal cells. PMID:22431514

Bdf1 Bromodomains Are Essential for Meiosis and the Expression of Meiotic-Specific Genes

PubMed Central

Perot, Jonathan; Arlotto, Marie; Mietton, Flore; Boland, Anne; Deleuze, Jean-François; Ferro, Myriam; Govin, Jérôme

2017-01-01

Bromodomain and Extra-terminal motif (BET) proteins play a central role in transcription regulation and chromatin signalling pathways. They are present in unicellular eukaryotes and in this study, the role of the BET protein Bdf1 has been explored in Saccharomyces cerevisiae. Mutation of Bdf1 bromodomains revealed defects on both the formation of spores and the meiotic progression, blocking cells at the exit from prophase, before the first meiotic division. This phenotype is associated with a massive deregulation of the transcription of meiotic genes and Bdf1 bromodomains are required for appropriate expression of the key meiotic transcription factor NDT80 and almost all the Ndt80-inducible genes, including APC complex components. Bdf1 notably accumulates on the promoter of Ndt80 and its recruitment is dependent on Bdf1 bromodomains. In addition, the ectopic expression of NDT80 during meiosis partially bypasses this dependency. Finally, purification of Bdf1 partners identified two independent complexes with Bdf2 or the SWR complex, neither of which was required to complete sporulation. Taken together, our results unveil a new role for Bdf1 –working independently from its predominant protein partners Bdf2 and the SWR1 complex–as a regulator of meiosis-specific genes. PMID:28068333

A novel transgenic chimaeric mouse system for the rapid functional evaluation of genes encoding secreted proteins

PubMed Central

Kakitani, Makoto; Oshima, Takeshi; Horikoshi, Kaori; Yoshitome, Tetsuo; Ueda, Akiko; Kajikawa, Miwa; Iba, Yumi; Ozone, Yoshinao; Ijima, Yuki; Yoshino, Tohko; Itoh, Mikiko; Seki, Sachiko; Aoki, Ayako; Ishihara, Toshie; Shionoya, Michiyo; Makino, Utako; Kitada, Rina; Ohguma, Atsuko; Ohta, Takami; Yoshida, Yoshimasa; Kudoh, Hiroe; Hanaoka, Kazunori; Sibuya, Kazunori; Ishida, Isao; Kakeda, Minoru; Yagi, Mikio; Yoneya, Takashi; Tomizuka, Kazuma

2005-01-01

A major challenge of the post-genomic era is the functional characterization of anonymous open reading frames (ORFs) identified by the Human Genome Project. In this context, there is a strong requirement for the development of technologies that enhance our ability to analyze gene functions at the level of the whole organism. Here, we describe a rapid and efficient procedure to generate transgenic chimaeric mice that continuously secrete a foreign protein into the systemic circulation. The transgene units were inserted into the genomic site adjacent to the endogenous immunoglobulin (Ig) κ locus by homologous recombination, using a modified mouse embryonic stem (ES) cell line that exhibits a high frequency of homologous recombination at the Igκ region. The resultant ES clones were injected into embryos derived from a B-cell-deficient host strain, thus producing chimaerism-independent, B-cell-specific transgene expression. This feature of the system eliminates the time-consuming breeding typically implemented in standard transgenic strategies and allows for evaluating the effect of ectopic transgene expression directly in the resulting chimaeric mice. To demonstrate the utility of this system we showed high-level protein expression in the sera and severe phenotypes in human EPO (hEPO) and murine thrombopoietin (mTPO) transgenic chimaeras. PMID:15914664

The neurite outgrowth inhibitor Nogo-A promotes denervation in an amyotrophic lateral sclerosis model

PubMed Central

Jokic, Natasa; Gonzalez de Aguilar, Jose-Luis; Dimou, Leda; Lin, Shuo; Fergani, Anissa; Ruegg, Markus A; Schwab, Martin E; Dupuis, Luc; Loeffler, Jean-Philippe

2006-01-01

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by motor neuron loss and muscle wasting. In muscles of ALS patients, Nogo-A—a protein known to inhibit axon regeneration—is ectopically expressed at levels that correlate with the severity of the clinical symptoms. We now show that the genetic ablation of Nogo-A extends survival and reduces muscle denervation in a mouse model of ALS. In turn, overexpression of Nogo-A in wild-type muscle fibres leads to shrinkage of the postsynapse and retraction of the presynaptic motor ending. This suggests that the expression of Nogo-A occurring early in ALS skeletal muscle could cause repulsion and destabilization of the motor nerve terminals, and subsequent dying back of the axons and motor neurons. PMID:17039253

Effects of an Antagonistic Analog of Growth Hormone-Releasing Hormone on Endometriosis in a Mouse Model and In Vitro.

PubMed

Köster, Frank; Jin, Li; Shen, Yuanming; Schally, Andrew V; Cai, Ren-Zhi; Block, Norman L; Hornung, Daniela; Marschner, Gabriele; Rody, Achim; Engel, Jörg B; Finas, Dominique

2017-11-01

Endometriosis is a benign gynecologic disorder causing dysmenorrhea, pelvic pain, and subfertility. Receptors for the growth hormone-releasing hormone (GHRH) were found in endometriotic tissues. Antagonists of GHRH have been used to inhibit the growth of endometriotic endometrial stromal cells. In this study, the GHRH receptor splice variant (SV) 1 was detected in human endometrial tissue samples by Western blots and quantitative reverse transcription polymerase chain reaction (qRT-PCR). The highest messenger RNA (mRNA) and protein levels of SV1 were found in eutopic endometrium from patients with endometriosis compared to ectopic endometriotic tissues and endometrium from normal patients. The highest expression for GHRH mRNA was found by qRT-PCR in ectopic endometriosis lesions. In an in vivo mouse model with human endometrial explants from patients with endometriosis, 10 μg MIA-602 per day resulted in significantly smaller human endometrial xenotransplants after 4 weeks compared to mice treated with vehicle. The endometrial tissues expressed SV1 before and after xenotransplantation. The proliferation of endometrial stromal cells as well as the endometriosis cell lines 12-Z and 49-Z was decreased by exposure to 1 μM MIA-602 after 72 hours. The protein levels of epithelial growth factor receptors in 12-Z and 49-Z cell lines were reduced 48 and 72 hours after the administration of 1 μM MIA-602. MIA-602 decreased the activation of the MAP-kinases ERK-1/2. Our study demonstrates the presence of SV1 receptor as a target for treatment with GHRH antagonist in endometriosis. Endometrial tissues respond to MIA-602 with inhibition of proliferation in vitro and in vivo. The use of MIA-602 could be an effective supplement to the treatment strategies in endometriosis.

3' Untranslated regions mediate transcriptional interference between convergent genes both locally and ectopically in Saccharomyces cerevisiae.

PubMed

Wang, Luwen; Jiang, Ning; Wang, Lin; Fang, Ou; Leach, Lindsey J; Hu, Xiaohua; Luo, Zewei

2014-01-01

Paired sense and antisense (S/AS) genes located in cis represent a structural feature common to the genomes of both prokaryotes and eukaryotes, and produce partially complementary transcripts. We used published genome and transcriptome sequence data and found that over 20% of genes (645 pairs) in the budding yeast Saccharomyces cerevisiae genome are arranged in convergent pairs with overlapping 3'-UTRs. Using published microarray transcriptome data from the standard laboratory strain of S. cerevisiae, our analysis revealed that expression levels of convergent pairs are significantly negatively correlated across a broad range of environments. This implies an important role for convergent genes in the regulation of gene expression, which may compensate for the absence of RNA-dependent mechanisms such as micro RNAs in budding yeast. We selected four representative convergent gene pairs and used expression assays in wild type yeast and its genetically modified strains to explore the underlying patterns of gene expression. Results showed that convergent genes are reciprocally regulated in yeast populations and in single cells, whereby an increase in expression of one gene produces a decrease in the expression of the other, and vice-versa. Time course analysis of the cell cycle illustrated the functional significance of this relationship for the three pairs with relevant functional roles. Furthermore, a series of genetic modifications revealed that the 3'-UTR sequence plays an essential causal role in mediating transcriptional interference, which requires neither the sequence of the open reading frame nor the translation of fully functional proteins. More importantly, transcriptional interference persisted even when one of the convergent genes was expressed ectopically (in trans) and therefore does not depend on the cis arrangement of convergent genes; we conclude that the mechanism of transcriptional interference cannot be explained by the transcriptional collision model, which postulates a clash between simultaneous transcriptional processes occurring on opposite DNA strands.

3′ Untranslated Regions Mediate Transcriptional Interference between Convergent Genes Both Locally and Ectopically in Saccharomyces cerevisiae

PubMed Central

Wang, Luwen; Jiang, Ning; Wang, Lin; Fang, Ou; Leach, Lindsey J.; Hu, Xiaohua; Luo, Zewei

2014-01-01

Paired sense and antisense (S/AS) genes located in cis represent a structural feature common to the genomes of both prokaryotes and eukaryotes, and produce partially complementary transcripts. We used published genome and transcriptome sequence data and found that over 20% of genes (645 pairs) in the budding yeast Saccharomyces cerevisiae genome are arranged in convergent pairs with overlapping 3′-UTRs. Using published microarray transcriptome data from the standard laboratory strain of S. cerevisiae, our analysis revealed that expression levels of convergent pairs are significantly negatively correlated across a broad range of environments. This implies an important role for convergent genes in the regulation of gene expression, which may compensate for the absence of RNA-dependent mechanisms such as micro RNAs in budding yeast. We selected four representative convergent gene pairs and used expression assays in wild type yeast and its genetically modified strains to explore the underlying patterns of gene expression. Results showed that convergent genes are reciprocally regulated in yeast populations and in single cells, whereby an increase in expression of one gene produces a decrease in the expression of the other, and vice-versa. Time course analysis of the cell cycle illustrated the functional significance of this relationship for the three pairs with relevant functional roles. Furthermore, a series of genetic modifications revealed that the 3′-UTR sequence plays an essential causal role in mediating transcriptional interference, which requires neither the sequence of the open reading frame nor the translation of fully functional proteins. More importantly, transcriptional interference persisted even when one of the convergent genes was expressed ectopically (in trans) and therefore does not depend on the cis arrangement of convergent genes; we conclude that the mechanism of transcriptional interference cannot be explained by the transcriptional collision model, which postulates a clash between simultaneous transcriptional processes occurring on opposite DNA strands. PMID:24465217

Hepatoma-derived growth factor: A survival-related protein in prostate oncogenesis and a potential target for vitamin K2.

PubMed

Shetty, Aditya; Dasari, Subramanyam; Banerjee, Souresh; Gheewala, Taher; Zheng, Guoxing; Chen, Aoshuang; Kajdacsy-Balla, Andre; Bosland, Maarten C; Munirathinam, Gnanasekar

2016-11-01

Hepatoma-derived growth factor (HDGF) is a heparin-binding growth factor, which has previously been shown to be expressed in a variety of cancers. HDGF overexpression has also previously been correlated with a poor prognosis in several cancers. The significance of HDGF in prostate cancer, however, has not been investigated. Here, we show that HDGF is overexpressed in both androgen-sensitive LNCaP cells and androgen-insensitive DU145, 22RV1, and PC-3 cells. Forced overexpression enhanced cell viability of RWPE-1 cells, whereas HDGF knockdown reduced cell proliferation in human prostate cancer cells. We also show that HDGF may serve as a survival-related protein as ectopic overexpression of HDGF in RWPE cells up-regulated the expression of antiapoptosis proteins cyclin E and BCL-2, whereas simultaneously down-regulating proapoptotic protein BAX. Western blot analysis also showed that HDGF overexpression modulated the activity of phospho-AKT as well as NF-kB, and these results correlated with in vitro migration and invasion assays. We next assessed the therapeutic potential of HDGF inhibition with a HDGF monoclonal antibody and vitamin k 2 , showing reduced cell proliferation as well as inhibition of NF-kB expression in HDGF overexpressed RWPE cells treated with a HDGF monoclonal antibody and vitamin K 2 . Collectively, our results suggest that HDGF is a relevant protein in prostate oncogenesis and may serve as a potential therapeutic target in prostate cancer. Copyright © 2016 Elsevier Inc. All rights reserved.

Hereditary mixed polyposis syndrome is caused by a 40-kb upstream duplication that leads to increased and ectopic expression of the BMP antagonist GREM1.

PubMed

Jaeger, Emma; Leedham, Simon; Lewis, Annabelle; Segditsas, Stefania; Becker, Martin; Cuadrado, Pedro Rodenas; Davis, Hayley; Kaur, Kulvinder; Heinimann, Karl; Howarth, Kimberley; East, James; Taylor, Jenny; Thomas, Huw; Tomlinson, Ian

2012-05-06

Hereditary mixed polyposis syndrome (HMPS) is characterized by apparent autosomal dominant inheritance of multiple types of colorectal polyp, with colorectal carcinoma occurring in a high proportion of affected individuals. Here, we use genetic mapping, copy-number analysis, exclusion of mutations by high-throughput sequencing, gene expression analysis and functional assays to show that HMPS is caused by a duplication spanning the 3' end of the SCG5 gene and a region upstream of the GREM1 locus. This unusual mutation is associated with increased allele-specific GREM1 expression. Whereas GREM1 is expressed in intestinal subepithelial myofibroblasts in controls, GREM1 is predominantly expressed in the epithelium of the large bowel in individuals with HMPS. The HMPS duplication contains predicted enhancer elements; some of these interact with the GREM1 promoter and can drive gene expression in vitro. Increased GREM1 expression is predicted to cause reduced bone morphogenetic protein (BMP) pathway activity, a mechanism that also underlies tumorigenesis in juvenile polyposis of the large bowel.

Exposure to tributyltin induces endoplasmic reticulum stress and the unfolded protein response in zebrafish.

PubMed

Komoike, Yuta; Matsuoka, Masato

2013-10-15

Tributyltin (TBT) is a major marine contaminant and causes endocrine disruption, hepatotoxicity, immunotoxicity, and neurotoxicity. However, the molecular mechanisms underlying the toxicity of TBT have not been fully elucidated. We examined whether exposure to TBT induces the endoplasmic reticulum (ER) stress response in zebrafish, a model organism. Zebrafish-derived BRF41 fibroblast cells were exposed to 0.5 or 1 μM TBT for 0.5-16 h and subsequently lysed and immunoblotted to detect ER stress-related proteins. Zebrafish embryos, grown until 32 h post fertilization (hpf), were exposed to 1 μM TBT for 16 h and used in whole mount in situ hybridization and immunohistochemistry to visualize the expression of ER chaperones and an ER stress-related apoptosis factor. Exposure of the BRF41 cells to TBT caused phosphorylation of the zebrafish homolog of protein kinase RNA-activated-like ER kinase (PERK), eukaryotic translation initiation factor 2 alpha (eIF2α), and inositol-requiring enzyme 1 (IRE1), characteristic splicing of X-box binding protein 1 (XBP1) mRNA, and enhanced expression of activating transcription factor 4 (ATF4) protein. In TBT-exposed zebrafish embryos, ectopic expression of the gene encoding zebrafish homolog of the 78 kDa glucose-regulating protein (GRP78) and gene encoding CCAAT/enhancer-binding protein homologous protein (CHOP) was detected in the precursors of the neuromast, which is a sensory organ for detecting water flow and vibration. Our in vitro and in vivo studies revealed that exposure of zebrafish to TBT induces the ER stress response via activation of both the PERK-eIF2α and IRE1-XBP1 pathways of the unfolded protein response (UPR) in an organ-specific manner. Copyright © 2013 Elsevier B.V. All rights reserved.

The class I protein AtTCP15 modulates plant development through a pathway that overlaps with the one affected by CIN-like TCP proteins.

PubMed

Uberti-Manassero, Nora G; Lucero, Leandro E; Viola, Ivana L; Vegetti, Abelardo C; Gonzalez, Daniel H

2012-01-01

The function of the class I TCP transcription factor TCP15 from Arabidopsis thaliana has been studied through the analysis of plants that express a fusion of this protein to the EAR repressor domain. Constitutive expression of TCP15-EAR produces growth arrest at the seedling stage, before leaf emergence. Expression of the repressor fusion from the AtTCP15 promoter produces small plants with leaves whose margins progressively curve upwards, starting from the basal part of the lamina. Leaves contain smaller and less differentiated cells, both on the adaxial and abaxial sides. The abaxial domain is relatively enlarged, with disorganized cells separated by empty spaces. TCP15-EAR also affects the growth of leaf petioles, flower pedicels, and anther filaments. Flowers show reduced elongation of the three outer whorls and altered gynoecia with irregular carpel surfaces and enlarged repla. Ectopic stigma-like structures develop from medial and basal parts of the replum. TCP15-EAR produces an increase in expression of the boundary-specific genes LOB, CUC1, and CUC2. Changes in CUC1 and CUC2 expression can be explained by the existence of lower levels of miR164 in leaves and the repression of IAA3/SHY2 and the SAUR-like gene At1g29460 in leaves and flowers. TCP15 binds to the promoter regions of IAA3/SHY2 and At1g29460, suggesting that these genes may be direct targets of the transcription factor. The results indicate that TCP15 regulates the expression of boundary-specific genes through a pathway that affects auxin homeostasis and partially overlaps with the one modulated by class II CIN-like TCP proteins.

Ectopic expression of syncollin in INS-1 beta-cells sorts it into granules and impairs regulated secretion.

PubMed

Li, Jingsong; Luo, Ruihua; Hooi, Shing Chuan; Ruga, Pilar; Zhang, Jiping; Meda, Paolo; Li, GuoDong

2005-03-22

Syncollin was first demonstrated to be a protein capable of affecting granule fusion in a cell-free system, but later studies revealed its luminal localization in zymogen granules. To determine its possible role in exocytosis in the intact cell, syncollin and a truncated form of the protein (lacking the N-terminal hydrophobic domain) were stably transfected in insulin-secreting INS-1 cells since these well-studied exocytotic cells appear not to express the protein per se. Studies by subcellular fractionation analysis, double immunofluorescence staining, and electron microscopy examination revealed that transfection of syncollin produced strong signals in the insulin secretory granules, whereas the product from transfecting the truncated syncollin was predominantly associated with the Golgi apparatus and to a lesser degree with the endoplasmic reticulum. The expressed products were associated with membranes and not the soluble fractions in either cytoplasm or the lumens of organelles. Importantly, insulin release stimulated by various secretagogues was severely impaired in cells expressing syncollin, but not affected by expressing truncated syncollin. Transfection of syncollin appeared not to impede insulin biosynthesis and processing, since cellular contents of proinsulin and insulin and the number of secretory granules were not altered. In addition, the early signals (membrane depolarization and Ca(2+) responses) for regulated insulin secretion were unaffected. These findings indicate that syncollin may be targeted to insulin secretory granules specifically and impair regulated secretion at a distal stage.

Regulation of protease-activated receptor 1 signaling by the adaptor protein complex 2 and R4 subfamily of regulator of G protein signaling proteins.

PubMed

Chen, Buxin; Siderovski, David P; Neubig, Richard R; Lawson, Mark A; Trejo, Joann

2014-01-17

The G protein-coupled protease-activated receptor 1 (PAR1) is irreversibly proteolytically activated by thrombin. Hence, the precise regulation of PAR1 signaling is important for proper cellular responses. In addition to desensitization, internalization and lysosomal sorting of activated PAR1 are critical for the termination of signaling. Unlike most G protein-coupled receptors, PAR1 internalization is mediated by the clathrin adaptor protein complex 2 (AP-2) and epsin-1, rather than β-arrestins. However, the function of AP-2 and epsin-1 in the regulation of PAR1 signaling is not known. Here, we report that AP-2, and not epsin-1, regulates activated PAR1-stimulated phosphoinositide hydrolysis via two different mechanisms that involve, in part, a subset of R4 subfamily of "regulator of G protein signaling" (RGS) proteins. A significantly greater increase in activated PAR1 signaling was observed in cells depleted of AP-2 using siRNA or in cells expressing a PAR1 (420)AKKAA(424) mutant with defective AP-2 binding. This effect was attributed to AP-2 modulation of PAR1 surface expression and efficiency of G protein coupling. We further found that ectopic expression of R4 subfamily members RGS2, RGS3, RGS4, and RGS5 reduced activated PAR1 wild-type signaling, whereas signaling by the PAR1 AKKAA mutant was minimally affected. Intriguingly, siRNA-mediated depletion analysis revealed a function for RGS5 in the regulation of signaling by the PAR1 wild type but not the AKKAA mutant. Moreover, activation of the PAR1 wild type, and not the AKKAA mutant, induced Gαq association with RGS3 via an AP-2-dependent mechanism. Thus, AP-2 regulates activated PAR1 signaling by altering receptor surface expression and through recruitment of RGS proteins.

Downregulation of the c-Fes protein-tyrosine kinase inhibits the proliferation of human renal carcinoma cells

PubMed Central

Kanda, Shigeru; Miyata, Yasuyoshi; Kanetake, Hiroshi; Smithgall, Thomas E.

2009-01-01

The c-Fes protein-tyrosine kinase is associated with growth and differentiation of hematopoietic, neuronal, vascular endothelial and epithelial cell types. In this study, we investigated whether small interfering RNA (siRNA)-mediated knockdown of c-Fes expression affected proliferation of the human renal carcinoma cell lines, ACHN and VMRC-RCW. Immunofluorescence microscopy showed that c-Fes was expressed in both the cytosol and nuclei of these cells, and siRNA treatment preferentially downregulated c-Fes expression in the cytosol. Knock-down of c-Fes inhibited cellular proliferation in a dose-dependent manner with minimal increase in cell death. c-Fes siRNA treatment also downregulated the phosphorylation of Akt1 on S473 and IKKα on T23, and cyclin D1 expression, enhanced the expression of IκBα, and prevented the nuclear localization of NFκB. Treatment with an NFκB inhibitory peptide (SN50) also blocked the proliferation and nuclear localization of NFκB in these cells. The effect of SN50 treatment was not enhanced by c-Fes siRNA, suggesting that downregulation of c-Fes expression inhibited cell cycle progression through the Akt1/NFκB pathway. In contrast to siRNA-mediated knockdown, ectopic expression of either wild-type or kinase-inactive c-Fes in renal carcinoma cells failed to alter their proliferation in vitro and in vivo. Thus, suppression of proliferation resulting from siRNA-mediated knockdown may depend upon an expression of c-Fes protein rather than its kinase activity. Taken together, our results indicate that downregulation of c-Fes expression may be a potential therapeutic strategy for advanced human renal cell carcinoma and inhibition of its kinase activity as an antiangiogenic therapy does not seem to induce the growth of human renal carcinoma cells. PMID:19082481

Dynamic localization of MreB in Vibrio parahaemolyticus and in the ectopic host bacterium Escherichia coli.

PubMed

Chiu, Shen-Wen; Chen, Shau-Yan; Wong, Hin-chung

2008-11-01

MreB, a homolog of eukaryotic actin, participates in morphogenesis, cell division, cell polarity, and chromosome segregation in prokaryotes. In this study, a yellow fluorescent protein conjugate (YFP-MreB(Vp)) was generated to investigate the behavior of MreB in merodiploid strain SC9 of the enteropathogen Vibrio parahaemolyticus. Under normal growth conditions, YFP-MreB(Vp) formed helical filaments with a pitch of 0.64 +/- 0.09 microm in about 85% of exponential-phase cells, and different clusters, relaxed coils, and ring configurations were observed in a small proportion of the cells. Overexpression of YFP-MreB(Vp) substantially altered the structure of the MreB cytoskeleton and resulted in swollen and pleomorphic cells. Disturbing the activities of penicillin-binding proteins or adding magnesium suppressed the morphological distortions. These results indicate that mislocalization of cell wall-synthesizing machinery was responsible for morphological abnormality. By expressing YFP-MreB(Vp) in the ectopic host bacterium Escherichia coli, shrinkage, fragmentation, and annealing of MreB(Vp) filaments were directly observed. This work revealed the dynamic pattern of the localization of YFP-MreB(Vp) in V. parahaemolyticus and its relationship to cell morphogenesis, and the YFP-MreB(Vp)-E. coli system may be used to investigate the dynamic spatial structures of the MreB cytoskeleton in vivo.

Genome-wide expressions in autologous eutopic and ectopic endometrium of fertile women with endometriosis.

PubMed

Khan, Meraj A; Sengupta, Jayasree; Mittal, Suneeta; Ghosh, Debabrata

2012-09-24

In order to obtain a lead of the pathophysiology of endometriosis, genome-wide expressional analyses of eutopic and ectopic endometrium have earlier been reported, however, the effects of stages of severity and phases of menstrual cycle on expressional profiles have not been examined. The effect of genetic heterogeneity and fertility history on transcriptional activity was also not considered. In the present study, a genome-wide expression analysis of autologous, paired eutopic and ectopic endometrial samples obtained from fertile women (n=18) suffering from moderate (stage 3; n=8) or severe (stage 4; n=10) ovarian endometriosis during proliferative (n=13) and secretory (n=5) phases of menstrual cycle was performed. Individual pure RNA samples were subjected to Agilent's Whole Human Genome 44K microarray experiments. Microarray data were validated (P<0.01) by estimating transcript copy numbers by performing real time RT-PCR of seven (7) arbitrarily selected genes in all samples. The data obtained were subjected to differential expression (DE) and differential co-expression (DC) analyses followed by networks and enrichment analysis, and gene set enrichment analysis (GSEA). The reproducibility of prediction based on GSEA implementation of DC results was assessed by examining the relative expressions of twenty eight (28) selected genes in RNA samples obtained from fresh pool of eutopic and ectopic samples from confirmed ovarian endometriosis patients with stages 3 and 4 (n=4/each) during proliferative and secretory (n=4/each) phases. Higher clustering effect of pairing (cluster distance, cd=0.1) in samples from same individuals on expressional arrays among eutopic and ectopic samples was observed as compared to that of clinical stages of severity (cd=0.5) and phases of menstrual cycle (cd=0.6). Post hoc analysis revealed anomaly in the expressional profiles of several genes associated with immunological, neuracrine and endocrine functions and gynecological cancers however with no overt oncogenic potential in endometriotic tissue. Dys-regulation of three (CLOCK, ESR1, and MYC) major transcription factors appeared to be significant causative factors in the pathogenesis of ovarian endometriosis. A novel cohort of twenty-eight (28) genes representing potential marker for ovarian endometriosis in fertile women was discovered. Dysfunctional expression of immuno-neuro-endocrine behaviour in endometrium appeared critical to endometriosis. Although no overt oncogenic potential was evident, several genes associated with gynecological cancers were observed to be high in the expressional profiles in endometriotic tissue.

Ectopic Activation of Wnt/β-Catenin Signaling in Lens Fiber Cells Results in Cataract Formation and Aberrant Fiber Cell Differentiation

PubMed Central

Antosova, Barbora; Smolikova, Jana; Borkovcova, Romana; Strnad, Hynek; Lachova, Jitka; Machon, Ondrej; Kozmik, Zbynek

2013-01-01

The Wnt/β-catenin signaling pathway controls many processes during development, including cell proliferation, cell differentiation and tissue homeostasis, and its aberrant regulation has been linked to various pathologies. In this study we investigated the effect of ectopic activation of Wnt/β-catenin signaling during lens fiber cell differentiation. To activate Wnt/β-catenin signaling in lens fiber cells, the transgenic mouse referred to as αA-CLEF was generated, in which the transactivation domain of β-catenin was fused to the DNA-binding protein LEF1, and expression of the transgene was controlled by αA-crystallin promoter. Constitutive activation of Wnt/β-catenin signaling in lens fiber cells of αA-CLEF mice resulted in abnormal and delayed fiber cell differentiation. Moreover, adult αA-CLEF mice developed cataract, microphthalmia and manifested downregulated levels of γ-crystallins in lenses. We provide evidence of aberrant expression of cell cycle regulators in embryonic lenses of αA-CLEF transgenic mice resulting in the delay in cell cycle exit and in the shift of fiber cell differentiation to the central fiber cell compartment. Our results indicate that precise regulation of the Wnt/β-catenin signaling activity during later stages of lens development is essential for proper lens fiber cell differentiation and lens transparency. PMID:24205179

Targeted Ablation of the Abcc6 Gene Results in Ectopic Mineralization of Connective Tissues

PubMed Central

Klement, John F.; Matsuzaki, Yasushi; Jiang, Qiu-Jie; Terlizzi, Joseph; Choi, Hae Young; Fujimoto, Norihiro; Li, Kehua; Pulkkinen, Leena; Birk, David E.; Sundberg, John P.; Uitto, Jouni

2005-01-01

Pseudoxanthoma elasticum (PXE), characterized by connective tissue mineralization of the skin, eyes, and cardiovascular system, is caused by mutations in the ABCC6 gene. ABCC6 encodes multidrug resistance-associated protein 6 (MRP6), which is expressed primarily in the liver and kidneys. Mechanisms producing ectopic mineralization as a result of these mutations remain unclear. To elucidate this complex disease, a transgenic mouse was generated by targeted ablation of the mouse Abcc6 gene. Abcc6 null mice were negative for Mrp6 expression in the liver, and complete necropsies revealed profound mineralization of several tissues, including skin, arterial blood vessels, and retina, while heterozygous animals were indistinguishable from the wild-type mice. Particularly striking was the mineralization of vibrissae, as confirmed by von Kossa and alizarin red stains. Electron microscopy revealed mineralization affecting both elastic structures and collagen fibers. Mineralization of vibrissae was noted as early as 5 weeks of age and was progressive with age in Abcc6−/− mice but was not observed in Abcc6+/− or Abcc6+/+ mice up to 2 years of age. A total body computerized tomography scan of Abcc6−/− mice revealed mineralization in skin and subcutaneous tissue as well as in the kidneys. These data demonstrate aberrant mineralization of soft tissues in PXE-affected organs, and, consequently, these mice recapitulate features of this complex disease. PMID:16135817

The retrovirus HTLV-1 inserts an ectopic CTCF-binding site into the human genome.

PubMed

Satou, Yorifumi; Miyazato, Paola; Ishihara, Ko; Yaguchi, Hiroko; Melamed, Anat; Miura, Michi; Fukuda, Asami; Nosaka, Kisato; Watanabe, Takehisa; Rowan, Aileen G; Nakao, Mitsuyoshi; Bangham, Charles R M

2016-03-15

Human T-lymphotropic virus type 1 (HTLV-1) is a retrovirus that causes malignant and inflammatory diseases in ∼10% of infected people. A typical host has between 10(4) and 10(5) clones of HTLV-1-infected T lymphocytes, each clone distinguished by the genomic integration site of the single-copy HTLV-1 provirus. The HTLV-1 bZIP (HBZ) factor gene is constitutively expressed from the minus strand of the provirus, whereas plus-strand expression, required for viral propagation to uninfected cells, is suppressed or intermittent in vivo, allowing escape from host immune surveillance. It remains unknown what regulates this pattern of proviral transcription and latency. Here, we show that CTCF, a key regulator of chromatin structure and function, binds to the provirus at a sharp border in epigenetic modifications in the pX region of the HTLV-1 provirus in T cells naturally infected with HTLV-1. CTCF is a zinc-finger protein that binds to an insulator region in genomic DNA and plays a fundamental role in controlling higher order chromatin structure and gene expression in vertebrate cells. We show that CTCF bound to HTLV-1 acts as an enhancer blocker, regulates HTLV-1 mRNA splicing, and forms long-distance interactions with flanking host chromatin. CTCF-binding sites (CTCF-BSs) have been propagated throughout the genome by transposons in certain primate lineages, but CTCF binding has not previously been described in present-day exogenous retroviruses. The presence of an ectopic CTCF-BS introduced by the retrovirus in tens of thousands of genomic locations has the potential to cause widespread abnormalities in host cell chromatin structure and gene expression.

Conifer reproductive development involves B-type MADS-box genes with distinct and different activities in male organ primordia.

PubMed

Sundström, Jens; Engström, Peter

2002-07-01

The Norway spruce MADS-box genes DAL11, DAL12 and DAL13 are phylogenetically related to the angiosperm B-function MADS-box genes: genes that act together with A-function genes in specifying petal identity and with C-function genes in specifying stamen identity to floral organs. In this report we present evidence to suggest that the B-gene function in the specification of identity of the pollen-bearing organs has been conserved between conifers and angiosperms. Expression of DAL11 or DAL12 in transgenic Arabidopsis causes phenotypic changes which partly resemble those caused by ectopic expression of the endogenous B-genes. In similar experiments, flowers of Arabidopsis plants expressing DAL13 showed a different homeotic change in that they formed ectopic anthers in whorls one, two or four. We also demonstrate the capacity of the spruce gene products to form homodimers, and that DAL11 and DAL13 may form heterodimers with each other and with the Arabidopsis B-protein AP3, but not with PI, the second B-gene product in Arabidopsis. In situ hybridization experiments show that the conifer B-like genes are expressed specifically in developing pollen cones, but differ in both temporal and spatial distribution patterns. These results suggest that the B-function in conifers is dual and is separated into a meristem identity and an organ identity function, the latter function possibly being independent of an interaction with the C-function. Thus, even though an ancestral B-function may have acted in combination with C to specify micro- and megasporangia, the B-function has evolved differently in conifers and angiosperms.

Ectopic Overexpression of SlHsfA3, a Heat Stress Transcription Factor from Tomato, Confers Increased Thermotolerance and Salt Hypersensitivity in Germination in Transgenic Arabidopsis

PubMed Central

Li, Zhenjun; Zhang, Lili; Wang, Aoxue; Xu, Xiangyang; Li, Jingfu

2013-01-01

Plant heat stress transcription factors (Hsfs) are the critical components involved in mediating responses to various environmental stressors. However, the detailed roles of many plant Hsfs are far from fully understood. In this study, an Hsf (SlHsfA3) was isolated from the cultivated tomato (Solanum lycopersicum, Sl) and functionally characterized at the genetic and developmental levels. The nucleus-localized SlHsfA3 was basally and ubiquitously expressed in different plant organs. The expression of SlHsfA3 was induced dramatically by heat stress, moderately by high salinity, and slightly by drought, but was not induced by abscisic acid (ABA). The ectopic overexpression of SlHsfA3 conferred increased thermotolerance and late flowering phenotype to transgenic Arabidopsis plants. Moreover, SlHsfA3 played a negative role in controlling seed germination under salt stress. RNA-sequencing data demonstrated that a number of heat shock proteins (Hsps) and stress-associated genes were induced in Arabidopsis plants overexpressing SlHsfA3. A gel shift experiment and transient expression assays in Nicotiana benthamiana leaves demonstrated that SlHsfA3 directly activates the expression of SlHsp26.1-P and SlHsp21.5-ER. Taken together, our results suggest that SlHsfA3 behaves as a typical Hsf to contribute to plant thermotolerance. The late flowering and seed germination phenotypes and the RNA-seq data derived from SlHsfA3 overexpression lines lend more credence to the hypothesis that plant Hsfs participate in diverse physiological and biochemical processes related to adverse conditions. PMID:23349984

Complex relationship between mismatch repair proteins and MBD4 during immunoglobulin class switch recombination.

PubMed

Grigera, Fernando; Bellacosa, Alfonso; Kenter, Amy L

2013-01-01

Mismatch repair (MMR) safeguards against genomic instability and is required for efficient Ig class switch recombination (CSR). Methyl CpG binding domain protein 4 (MBD4) binds to MutL homologue 1 (MLH1) and controls the post-transcriptional level of several MMR proteins, including MutS homologue 2 (MSH2). We show that in WT B cells activated for CSR, MBD4 is induced and interacts with MMR proteins, thereby implying a role for MBD4 in CSR. However, CSR is in the normal range in Mbd4 deficient mice deleted for exons 2-5 despite concomitant reduction of MSH2. We show by comparison in Msh2(+/-) B cells that a two-fold reduction of MSH2 and MBD4 proteins is correlated with impaired CSR. It is therefore surprising that CSR occurs at normal frequencies in the Mbd4 deficient B cells where MSH2 is reduced. We find that a variant Mbd4 transcript spanning exons 1,6-8 is expressed in Mbd4 deficient B cells. This transcript can be ectopically expressed and produces a truncated MBD4 peptide. Thus, the 3' end of the Mbd4 locus is not silent in Mbd4 deficient B cells and may contribute to CSR. Our findings highlight a complex relationship between MBD4 and MMR proteins in B cells and a potential reconsideration of their role in CSR.

Isolation and characterization of two VpYABBY genes from wild Chinese Vitis pseudoreticulata.

PubMed

Xiang, J; Liu, R Q; Li, T M; Han, L J; Zou, Y; Xu, T F; Wei, J Y; Wang, Y J; Xu, Y

2013-12-01

The establishment of abaxial-adaxial polarity is an important feature of the development of lateral organs in plants. Members of the YABBY gene family may be specific to seed-plant-specific transcriptional regulators that play critical roles in promoting abaxial cell fate in the model eudicot, Arabidopsis thaliana. However, recent study has shown that the roles of YABBY genes are not conserved in the development of angiosperms. The establishment of abaxial-adaxial polarity has not been studied in perennial fruit crops. Grapes are an important fruit crop in many regions of the world. Investigating YABBY genes in grapevines should help us to discover more about the key genetic and molecular pathways in grapevine development. To understand the characterization of YABBY genes in grapevines, two YABBY genes, VpYABBY1 (GenBank accession No. KC139089) and VpYABBY2 (GenBank accession No. KC139090), were isolated from the wild Chinese species Vitis pseudoreticulata. Both of these encode YABBY proteins. Sequence characterization and phylogenetic analyses show that VpYABBY1 is group classified into the FIL subfamily while VpYABBY2 is a member of the YAB2 subfamily of Arabidopsis thaliana. Subcellular localization analysis indicates that VpYABBY1 and VpYABBY2 proteins are localized in the nucleus. Tissue specific expressional analysis reveals that VpYABBY1 is expressed strongly in young leaves of grape but only weakly in the mature leaves. Meanwhile, VpYABBY2 is expressed in grape stems, flowers, tendrils, and leaves. Transgenic Arabidopsis plants ectopically expressing VpYABBY1 caused the partial abaxialization of the adaxial epidermises of leaves, behaving similarly to those over-expressing FIL or YAB3 with abaxialized lateral organs. By contrast, ectopic expression of VpYABBY2 in Arabidopsis did not cause any alteration in the adaxial-abaxial polarity. Sequence characterization and phylogenetic analysis revealed that VpYABBY1 and VpYABBY2 are group-classified into two different subfamilies. They have diverged functionally in the control of lateral organ development. VpYABBY1 may have a function in leaf development, while VpYABBY2 may play a specific role in carpel development and grape berry morphogenesis. It is further possible that during the evolution of different species, YABBY family members have preserved different expression regulatory systems and functions.

Ectopic Cdx2 Expression in Murine Esophagus Models an Intermediate Stage in the Emergence of Barrett's Esophagus

PubMed Central

Kong, Jianping; Crissey, Mary Ann; Funakoshi, Shinsuke; Kreindler, James L.; Lynch, John P.

2011-01-01

Barrett's esophagus (BE) is an intestinal metaplasia that occurs in the setting of chronic acid and bile reflux and is associated with a risk for adenocarcinoma. Expression of intestine-specific transcription factors in the esophagus likely contributes to metaplasia development. Our objective was to explore the effects of an intestine-specific transcription factor when expressed in the mouse esophageal epithelium. Transgenic mice were derived in which the transcription factor Cdx2 is expressed in squamous epithelium using the murine Keratin-14 gene promoter. Effects of the transgene upon cell proliferation and differentiation, gene expression, and barrier integrity were explored. K14-Cdx2 mice express the Cdx2 transgene in esophageal squamous tissues. Cdx2 expression was associated with reduced basal epithelial cell proliferation and altered cell morphology. Ultrastructurally two changes were noted. Cdx2 expression was associated with dilated space between the basal cells and diminished cell-cell adhesion caused by reduced Desmocollin-3 mRNA and protein expression. This compromised epithelial barrier function, as the measured trans-epithelial electrical resistance (TEER) of the K14-Cdx2 epithelium was significantly reduced compared to controls (1189 Ohm*cm2 ±343.5 to 508 Ohm*cm2±92.48, p = 0.0532). Secondly, basal cells with features of a transitional cell type, intermediate between keratinocytes and columnar Barrett's epithelial cells, were observed. These cells had reduced keratin bundles and increased endoplasmic reticulum levels, suggesting the adoption of secretory-cell features. Moreover, at the ultrastructural level they resembled “Distinctive” cells associated with multilayered epithelium. Treatment of the K14-Cdx2 mice with 5′-Azacytidine elicited expression of BE-associated genes including Cdx1, Krt18, and Slc26a3/Dra, suggesting the phenotype could be advanced under certain conditions. We conclude that ectopic Cdx2 expression in keratinocytes alters cell proliferation, barrier function, and differentiation. These altered cells represent a transitional cell type between normal squamous and columnar BE cells. The K14-Cdx2 mice represent a useful model to study progression from squamous epithelium to BE. PMID:21494671

Ectopic Cdx2 expression in murine esophagus models an intermediate stage in the emergence of Barrett's esophagus.

PubMed

Kong, Jianping; Crissey, Mary Ann; Funakoshi, Shinsuke; Kreindler, James L; Lynch, John P

2011-04-06

Barrett's esophagus (BE) is an intestinal metaplasia that occurs in the setting of chronic acid and bile reflux and is associated with a risk for adenocarcinoma. Expression of intestine-specific transcription factors in the esophagus likely contributes to metaplasia development. Our objective was to explore the effects of an intestine-specific transcription factor when expressed in the mouse esophageal epithelium. Transgenic mice were derived in which the transcription factor Cdx2 is expressed in squamous epithelium using the murine Keratin-14 gene promoter. Effects of the transgene upon cell proliferation and differentiation, gene expression, and barrier integrity were explored. K14-Cdx2 mice express the Cdx2 transgene in esophageal squamous tissues. Cdx2 expression was associated with reduced basal epithelial cell proliferation and altered cell morphology. Ultrastructurally two changes were noted. Cdx2 expression was associated with dilated space between the basal cells and diminished cell-cell adhesion caused by reduced Desmocollin-3 mRNA and protein expression. This compromised epithelial barrier function, as the measured trans-epithelial electrical resistance (TEER) of the K14-Cdx2 epithelium was significantly reduced compared to controls (1189 Ohm*cm(2) ±343.5 to 508 Ohm*cm(2)±92.48, p = 0.0532). Secondly, basal cells with features of a transitional cell type, intermediate between keratinocytes and columnar Barrett's epithelial cells, were observed. These cells had reduced keratin bundles and increased endoplasmic reticulum levels, suggesting the adoption of secretory-cell features. Moreover, at the ultrastructural level they resembled "Distinctive" cells associated with multilayered epithelium. Treatment of the K14-Cdx2 mice with 5'-Azacytidine elicited expression of BE-associated genes including Cdx1, Krt18, and Slc26a3/Dra, suggesting the phenotype could be advanced under certain conditions. We conclude that ectopic Cdx2 expression in keratinocytes alters cell proliferation, barrier function, and differentiation. These altered cells represent a transitional cell type between normal squamous and columnar BE cells. The K14-Cdx2 mice represent a useful model to study progression from squamous epithelium to BE.

Cooperative transformation and coexpression of bovine papillomavirus type 1 E5 and E7 proteins.

PubMed

Bohl, J; Hull, B; Vande Pol, S B

2001-01-01

Productively infected bovine fibropapillomas were examined for bovine papillomavirus type 1 (BPV-1) E7 localization. BPV-1 E7 was observed in the cytoplasm of basal and lower spinous epithelial cells, coexpressed in the cytoplasm of basal cells with the E5 oncoprotein. E7 was also observed in nucleoli throughout the basal and spinous layers but not in the granular cell layer. Ectopic expression of E7 in cultured epithelial cells gave rise to localization similar to that seen in productive fibropapillomas, with cytoplasmic and nucleolar expression observed. Consistent with the coexpression of E7 and E5 in basal keratinocytes, BPV-1 E7 cooperated with E5 as well as E6 in an anchorage independence transformation assay. While E5 is expressed in both basal and superficial differentiating keratinocytes, BPV-1 E7 is only observed in basal and lower spinous epithelial cells. Therefore, BPV-1 E7 may serve to modulate the cellular response of basal epithelial cells to E5 expression.

Combinatorial delivery of small interfering RNAs reduces RNAi efficacy by selective incorporation into RISC

PubMed Central

Castanotto, Daniela; Sakurai, Kumi; Lingeman, Robert; Li, Haitang; Shively, Louise; Aagaard, Lars; Soifer, Harris; Gatignol, Anne; Riggs, Arthur; Rossi, John J.

2007-01-01

Despite the great potential of RNAi, ectopic expression of shRNA or siRNAs holds the inherent risk of competition for critical RNAi components, thus altering the regulatory functions of some cellular microRNAs. In addition, specific siRNA sequences can potentially hinder incorporation of other siRNAs when used in a combinatorial approach. We show that both synthetic siRNAs and expressed shRNAs compete against each other and with the endogenous microRNAs for transport and for incorporation into the RNA induced silencing complex (RISC). The same siRNA sequences do not display competition when expressed from a microRNA backbone. We also show that TAR RNA binding protein (TRBP) is one of the sensors for selection and incorporation of the guide sequence of interfering RNAs. These findings reveal that combinatorial siRNA approaches can be problematic and have important implications for the methodology of expression and use of therapeutic interfering RNAs. PMID:17660190

Expression of the voltage-sensing phosphatase gene in the chick embryonic tissues and in the adult cerebellum.

PubMed

Yamaguchi, Shinji; Aoki, Naoya; Kitajima, Takaaki; Okamura, Yasushi; Homma, Koichi J

2014-10-01

Voltage-sensing phosphatase (VSP) consists of a transmembrane voltage sensor domain (VSD) and the cytoplasmic domain with phosphoinositide-phosphatase activities. It operates as the voltage sensor and directly translates membrane potential into phosphoinositide turnover by coupling VSD to the cytoplasmic domain. VSPs are evolutionarily conserved from marine invertebrate up to humans. Recently, we demonstrated that ectopic expression of the chick ortholog of VSP, Gg-VSP, in a fibroblast cell line caused characteristic cell process outgrowths. Co-expression of chick PTEN suppressed such morphological change, suggesting that VSP regulates cell shape by increasing PI(3,4)P2. However, the in vivo function of Gg-VSP remains unclear. Here, we showed that in chick embryos Gg-VSP is expressed in the stomach, mesonephros, pharyngeal arch, limb bud, somites, floor plate of neural tube, and notochord. In addition, both Gg-VSP transcripts and the protein were found in the cerebellar Purkinje neurons. These findings provide an insight into the physiological functions of VSP.

Non-canonical TAF complexes regulate active promoters in human embryonic stem cells.

PubMed

Maston, Glenn A; Zhu, Lihua Julie; Chamberlain, Lynn; Lin, Ling; Fang, Minggang; Green, Michael R

2012-11-13

The general transcription factor TFIID comprises the TATA-box-binding protein (TBP) and approximately 14 TBP-associated factors (TAFs). Here we find, unexpectedly, that undifferentiated human embryonic stem cells (hESCs) contain only six TAFs (TAFs 2, 3, 5, 6, 7 and 11), whereas following differentiation all TAFs are expressed. Directed and global chromatin immunoprecipitation analyses reveal an unprecedented promoter occupancy pattern: most active genes are bound by only TAFs 3 and 5 along with TBP, whereas the remaining active genes are bound by TBP and all six hESC TAFs. Consistent with these results, hESCs contain a previously undescribed complex comprising TAFs 2, 6, 7, 11 and TBP. Altering the composition of hESC TAFs, either by depleting TAFs that are present or ectopically expressing TAFs that are absent, results in misregulated expression of pluripotency genes and induction of differentiation. Thus, the selective expression and use of TAFs underlies the ability of hESCs to self-renew.DOI:http://dx.doi.org/10.7554/eLife.00068.001.

THAP5 is a human cardiac-specific inhibitor of cell cycle that is cleaved by the proapoptotic Omi/HtrA2 protease during cell death.

PubMed

Balakrishnan, Meenakshi P; Cilenti, Lucia; Mashak, Zineb; Popat, Paiyal; Alnemri, Emad S; Zervos, Antonis S

2009-08-01

Omi/HtrA2 is a mitochondrial serine protease that has a dual function: while confined in the mitochondria, it promotes cell survival, but when released into the cytoplasm, it participates in caspase-dependent as well as caspase-independent cell death. To investigate the mechanism of Omi/HtrA2's function, we set out to isolate and characterize novel substrates for this protease. We have identified Thanatos-associated protein 5 (THAP5) as a specific interactor and substrate of Omi/HtrA2 in cells undergoing apoptosis. This protein is an uncharacterized member of the THAP family of proteins. THAP5 has a unique pattern of expression and is found predominantly in the human heart, although a very low expression is also seen in the human brain and muscle. THAP5 protein is localized in the nucleus and, when ectopically expressed, induces cell cycle arrest. During apoptosis, THAP5 protein is degraded, and this process can be blocked using a specific Omi/HtrA2 inhibitor, leading to reduced cell death. In patients with coronary artery disease, THAP5 protein levels substantially decrease in the myocardial infarction area, suggesting a potential role of this protein in human heart disease. This work identifies human THAP5 as a cardiac-specific nuclear protein that controls cell cycle progression. Furthermore, during apoptosis, THAP5 is cleaved and removed by the proapoptotic Omi/HtrA2 protease. Taken together, we provide evidence to support that THAP5 and its regulation by Omi/HtrA2 provide a new link between cell cycle control and apoptosis in cardiomyocytes.

Functional characterization of DnSIZ1, a SIZ/PIAS-type SUMO E3 ligase from Dendrobium.

PubMed

Liu, Feng; Wang, Xiao; Su, Mengying; Yu, Mengyuan; Zhang, Shengchun; Lai, Jianbin; Yang, Chengwei; Wang, Yaqin

2015-09-17

SUMOylation is an important post-translational modification of eukaryotic proteins that involves the reversible conjugation of a small ubiquitin-related modifier (SUMO) polypeptide to its specific protein substrates, thereby regulating numerous complex cellular processes. The PIAS (protein inhibitor of activated signal transducers and activators of transcription [STAT]) and SIZ (scaffold attachment factor A/B/acinus/PIAS [SAP] and MIZ) proteins are SUMO E3 ligases that modulate SUMO conjugation. The characteristic features and SUMOylation mechanisms of SIZ1 protein in monocotyledon are poorly understood. Here, we examined the functions of a homolog of Arabidopsis SIZ1, a functional SIZ/PIAS-type SUMO E3 ligase from Dendrobium. In Dendrobium, the predicted DnSIZ1 protein has domains that are highly conserved among SIZ/PIAS-type proteins. DnSIZ1 is widely expressed in Dendrobium organs and has a up-regulated trend by treatment with cold, high temperature and wounding. The DnSIZ1 protein localizes to the nucleus and shows SUMO E3 ligase activity when expressed in an Escherichia coli reconstitution system. Moreover, ectopic expression of DnSIZ1 in the Arabidopsis siz1-2 mutant partially complements several phenotypes and results in enhanced levels of SUMO conjugates in plants exposed to heat shock conditions. We observed that DnSIZ1 acts as a negative regulator of flowering transition which may be via a vernalization-induced pathway. In addition, ABA-hypersensitivity of siz1-2 seed germination can be partially suppressed by DnSIZ1. Our results suggest that DnSIZ1 is a functional homolog of the Arabidopsis SIZ1 with SUMO E3 ligase activity and may play an important role in the regulation of Dendrobium stress responses, flowering and development.

BMP-non-responsive Sca1+ CD73+ CD44+ mouse bone marrow derived osteoprogenitor cells respond to combination of VEGF and BMP-6 to display enhanced osteoblastic differentiation and ectopic bone formation.

PubMed

Madhu, Vedavathi; Li, Ching-Ju; Dighe, Abhijit S; Balian, Gary; Cui, Quanjun

2014-01-01

Clinical trials on fracture repair have challenged the effectiveness of bone morphogenetic proteins (BMPs) but suggest that delivery of mesenchymal stem cells (MSCs) might be beneficial. It has also been reported that BMPs could not increase mineralization in several MSCs populations, which adds ambiguity to the use of BMPs. However, an exogenous supply of MSCs combined with vascular endothelial growth factor (VEGF) and BMPs is reported to synergistically enhance fracture repair in animal models. To elucidate the mechanism of this synergy, we investigated the osteoblastic differentiation of cloned mouse bone marrow derived MSCs (D1 cells) in vitro in response to human recombinant proteins of VEGF, BMPs (-2, -4, -6, -9) and the combination of VEGF with BMP-6 (most potent BMP). We further investigated ectopic bone formation induced by MSCs pre-conditioned with VEGF, BMP-6 or both. No significant increase in mineralization, phosphorylation of Smads 1/5/8 and expression of the ALP, COL1A1 and osterix genes was observed upon addition of VEGF or BMPs alone to the cells in culture. The lack of CD105, Alk1 and Alk6 expression in D1 cells correlated with poor response to BMPs indicating that a greater care in the selection of MSCs is necessary. Interestingly, the combination of VEGF and BMP-6 significantly increased the expression of ALP, COL1A1 and osterix genes and D1 cells pre-conditioned with VEGF and BMP-6 induced greater bone formation in vivo than the non-conditioned control cells or the cells pre-conditioned with either VEGF or BMP-6 alone. This enhanced bone formation by MSCs correlated with higher CADM1 expression and OPG/RANKL ratio in the implants. Thus, combined action of VEGF and BMP on MSCs enhances osteoblastic differentiation of MSCs and increases their bone forming ability, which cannot be achieved through use of BMPs alone. This strategy can be effectively used for bone repair.

Leaf-Like Sepals Induced by Ectopic Expression of a SHORT VEGETATIVE PHASE (SVP)-Like MADS-Box Gene from the Basal Eudicot Epimedium sagittatum

PubMed Central

Li, Zhineng; Zeng, Shaohua; Li, Yanbang; Li, Mingyang; Souer, Erik

2016-01-01

Epimedium L. (Berberidaceae, Ranales), a perennial traditional Chinese medicinal herb, has become a new popular landscape plant for ground cover and pot culture in many countries based on its excellent ornamental characteristics and, distinctive and diverse floral morphology. However, little is known about the molecular genetics of flower development in Epimedium sagittatum. Here, we describe the characterization of EsSVP that encodes a protein sharing 68, 54, and 35% similarity with SVP, AGAMOUS-like 24 (AGL24) and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1) in Arabidopsis, respectively. Quantitative RT-PCR (qRT-PCR) indicated that EsSVP transcripts were principally found in petiole and leaf tissues, with little expression in roots and flowers and no in fruits. The highest EsSVP expression was observed in leaves. The flowering time of 35S::EsSVP in most Arabidopsis thaliana and in all petunia plants was not affected in both photoperiod conditions, but 35S::EsSVP 5# and 35S::EsSVP 1# Arabidopsis lines induced late and early flowering under long day (LD, 14 h light/10 h dark) and short day (SD, 10 h light/14 h dark) conditions, respectively. The 35S::EsSVP Arabidopsis produced extra secondary inflorescence or floral meristems in the axils of the leaf-like sepals with excrescent trichomes, and leaf-like sepals not able to enclose the inner three whorls completely. Moreover, almost all transgenic Arabidopsis plants showed persistent sepals around the completely matured fruits. Upon ectopic expression of 35S::EsSVP in Petunia W115, sepals were enlarged, sometimes to the size of leaves; corollas were greenish and did not fully open. These results suggest that EsSVP is involved in inflorescence meristem identity and flowering time regulation in some conditions. Although, the SVP homologs might have suffered functional diversification among diverse species between core and basal eudicots, the protein functions are conserved between Arabidopsis/Petunia and Epimedium. PMID:27733858

Arabidopsis Microtubule-Associated Protein MAP65-3 Cross-Links Antiparallel Microtubules toward Their Plus Ends in the Phragmoplast via Its Distinct C-Terminal Microtubule Binding Domain[W

PubMed Central

Ho, Chin-Min Kimmy; Lee, Yuh-Ru Julie; Kiyama, Lindsay D.; Dinesh-Kumar, Savithramma P.; Liu, Bo

2012-01-01

Plant cytokinesis is brought about by the phragmoplast, which contains an antiparallel microtubule (MT) array. The MT-associated protein MAP65-3 acts as an MT-bundling factor that specifically cross-links antiparallel MTs near their plus ends. MAP65 family proteins contain an N-terminal dimerization domain and C-terminal MT interaction domain. Compared with other MAP65 isoforms, MAP65-3 contains an extended C terminus. A MT binding site was discovered in the region between amino acids 496 and 588 and found to be essential for the organization of phragmoplast MTs. The frequent cytokinetic failure caused by loss of MAP65-3 was not rescued by ectopic expression of MAP65-1 under the control of the MAP65-3 promoter, indicating nonoverlapping functions between the two isoforms. In the presence of MAP65-3, however, ectopic MAP65-1 appeared in the phragmoplast midline. We show that MAP65-1 could acquire the function of MAP65-3 when the C terminus of MAP65-3, which contains the MT binding site, was grafted to it. Our results also show that MAP65-1 and MAP65-3 may share redundant functions in MT stabilization. Such a stabilization effect was likely brought about by MT binding and bundling. We conclude that MAP65-3 contains a distinct C-terminal MT binding site with a specific role in cross-linking antiparallel MTs toward their plus ends in the phragmoplast. PMID:22570443

Loss of HSulf-1 expression enhances tumorigenicity by inhibiting Bim expression in ovarian cancer.

PubMed

He, Xiaoping; Khurana, Ashwani; Roy, Debarshi; Kaufmann, Scott; Shridhar, Viji

2014-10-15

The expression of human Sulfatase1 (HSulf-1) is downregulated in the majority of primary ovarian cancer tumors, but the functional consequence of this downregulation remains unclear. Using two different shRNAs (Sh1 and Sh2), HSulf-1 expression was stably downregulated in ovarian cancer OV202 cells. We found that HSulf-1-deficient OV202 Sh1 and Sh2 cells formed colonies in soft agar. In contrast, nontargeting control (NTC) shRNA-transduced OV202 cells did not form any colonies. Moreover, subcutaneous injection of OV202 HSulf-1-deficient cells resulted in tumor formation in nude mice, whereas OV202 NTC cells did not. Also, ectopic expression of HSulf-1 in ovarian cancer SKOV3 cells significantly suppressed tumor growth in nude mice. Here, we show that HSulf-1-deficient OV202 cells have markedly decreased expression of proapoptotic Bim protein, which can be rescued by restoring HSulf-1 expression in OV202 Sh1 cells. Enhanced expression of HSulf-1 in HSulf-1-deficient SKOV3 cells resulted in increased Bim expression. Decreased Bim levels after loss of HSulf-1 were due to increased p-ERK, because inhibition of ERK activity with PD98059 resulted in increased Bim expression. However, treatment with a PI3 kinase/AKT inhibitor, LY294002, failed to show any change in Bim protein level. Importantly, rescuing Bim expression in HSulf-1 knockdown cells significantly retarded tumor growth in nude mice. Collectively, these results suggest that loss of HSulf-1 expression promotes tumorigenicity in ovarian cancer through regulating Bim expression. © 2014 UICC.

AGL61 interacts with AGL80 and is required for central cell development in Arabidopsis.

PubMed

Steffen, Joshua G; Kang, Il-Ho; Portereiko, Michael F; Lloyd, Alan; Drews, Gary N

2008-09-01

The central cell of the female gametophyte plays a role in pollen tube guidance and in regulating the initiation of endosperm development. Following fertilization, the central cell gives rise to the seed's endosperm, which nourishes the developing embryo within the seed. The molecular mechanisms controlling specification and differentiation of the central cell are poorly understood. We identified AGL61 in a screen for transcription factor genes expressed in the female gametophyte. AGL61 encodes a Type I MADS domain protein, which likely functions as a transcription factor. Consistent with this, an AGL61-green fluorescent protein fusion protein is localized to the nucleus. In the context of the ovule and seed, AGL61 is expressed exclusively in the central cell and early endosperm. agl61 female gametophytes are affected in the central cell specifically. The morphological defects include an overall reduction in size of the central cell and a reduced or absent central cell vacuole. When fertilized with wild-type pollen, agl61 central cells fail to give rise to endosperm. In addition, synergid- and antipodal-expressed genes are ectopically expressed in agl61 central cells. The expression pattern and mutant phenotype of AGL61 are similar to those of AGL80, suggesting that AGL61 may function as a heterodimer with AGL80 within the central cell; consistent with this, AGL61 and AGL80 interact in yeast two-hybrid assays. Together, these data suggest that AGL61 functions as a transcription factor and controls the expression of downstream genes during central cell development.

Peripheral myelin protein-22 (PMP22) modulates alpha 6 integrin expression in the human endometrium.

PubMed

Rao, Rajiv G; Sudhakar, Deepthi; Hogue, Claire P; Amici, Stephanie; Gordon, Lynn K; Braun, Jonathan; Notterpek, Lucia; Goodglick, Lee; Wadehra, Madhuri

2011-04-25

PMP22, a member of the GAS3 family of tetraspan proteins, is associated with a variety of neurological diseases. Previous studies have shown that PMP22 is expressed in proliferative endometrium, but its function within this tissue is poorly understood. In this study, we first characterized the expression of PMP22 in the human menstrual cycle and began to characterize its function in the endometrium. Using a combination of immunohistochemistry and quantitative PCR, we characterized the expression of PMP22 in both proliferative and secretory endometrium. Differences in PMP22 expression between proliferative and secretory endometrium were determined using a Mann-Whitney U test. In order to investigate the influence of PMP22 on α6 integrin expression, cells were created that ectopically overexpressed PMP22 or expressed a siRNA to inhibit its expression. These cells were analyzed for changes in integrins and binding to extracellular matrices. In this study, we show that PMP22 expression is higher in proliferative phase than secretory phase. Functionally, we have begun to characterize the functional significance of this expression. Previous studies have suggested a link between PMP22 and α6 integrin, and therefore we asked whether PMP22 could associate or potentially modulate the expression of α6 integrin. Expression of both PMP22 and α6 integrin were detectable in endometrial epithelial and stromal cells, and we show that both proteins can associate and colocalize with each other. To understand if PMP22 directly altered the expression of a6 integrin, we examined cell lines with modulated levels of the protein. Overexpression of PMP22 was sufficient to increase α6 integrin surface expression with a concominant increase in binding to the extracellular matrix laminin, while a reduction in PMP22 suppressed α6 integrin surface expression. These findings suggest a physiologic role for PMP22 on the expression of α6 integrin. We predict that this may be important for the maintainence of endometrial integrity and to the disease biology associated with altered levels of α6 integrin expression in the endometrium.

MUC4 modulates human glioblastoma cell proliferation and invasion by upregulating EGFR expression.

PubMed

Li, Weihua; Wu, Chunming; Yao, Yiqun; Dong, Bin; Wei, Zhenqing; Lv, Xiupeng; Zhang, Jian; Xu, Yinghui

2014-04-30

Glioblastoma (GBM), the most common primary brain tumor, is the leading cause of deaths related to tumors in the central nervous system. The prognosis of GBM patients is currently poor, and the mechanisms underlying GBM genesis remain unclear. The expression of MUC4, a high-molecular-weight and highly glycosylated protein, has been studied in many cancers. However, information on MUC4 expression in GBM is limited. In this study, we found that MUC4 was overexpressed in GBM cell lines and tissues. The proliferation and invasive potential of GBM cells were significantly increased by the ectopic expression of MUC4. By contrast, RNA interference targeting MUC4 in GBM cells significantly decreased the proliferation and invasive potential of GBM cells. We also found that the expression of epidermal growth factor receptor (EGFR) was modulated by MUC4. EGFR inhibition by siRNA reversed the MUC4-induced proliferation and invasion. These results indicated that MUC4 expression in GBM was important in GBM cell proliferation and invasion, which may be partly associated with EGFR overexpression. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

A Gata2-Dependent Transcription Network Regulates Uterine Progesterone Responsiveness and Endometrial Function.

PubMed

Rubel, Cory A; Wu, San-Pin; Lin, Lin; Wang, Tianyuan; Lanz, Rainer B; Li, Xilong; Kommagani, Ramakrishna; Franco, Heather L; Camper, Sally A; Tong, Qiang; Jeong, Jae-Wook; Lydon, John P; DeMayo, Francesco J

2016-10-25

Altered progesterone responsiveness leads to female infertility and cancer, but underlying mechanisms remain unclear. Mice with uterine-specific ablation of GATA binding protein 2 (Gata2) are infertile, showing failures in embryo implantation, endometrial decidualization, and uninhibited estrogen signaling. Gata2 deficiency results in reduced progesterone receptor (PGR) expression and attenuated progesterone signaling, as evidenced by genome-wide expression profiling and chromatin immunoprecipitation. GATA2 not only occupies at and promotes expression of the Pgr gene but also regulates downstream progesterone responsive genes in conjunction with the PGR. Additionally, Gata2 knockout uteri exhibit abnormal luminal epithelia with ectopic TRP63 expressing squamous cells and a cancer-related molecular profile in a progesterone-independent manner. Lastly, we found a conserved GATA2-PGR regulatory network in both human and mice based on gene signature and path analyses using gene expression profiles of human endometrial tissues. In conclusion, uterine Gata2 regulates a key regulatory network of gene expression for progesterone signaling at the early pregnancy stage. Published by Elsevier Inc.

Epigenetic Repression of Matrix Metalloproteinases in Myofibroblastic Hepatic Stellate Cells through Histone Deacetylases 4

PubMed Central

Qin, Lan; Han, Yuan-Ping

2010-01-01

Matrix metalloproteinases (MMPs), which are highly expressed in acute injury, are progressively repressed or silenced in fibrotic liver, favoring extracellular matrix accumulation, while the underlying mechanism is largely unknown. Similarly, normal/quiescent hepatic stellate cells (HSCs) express high levels of MMPs in response to injury signals, such as interleukin-1. After transdifferentiation, the myofibroblastic HSCs are incapable of expressing many MMPs; however, the major signaling pathways required for MMP expression are intact, indicating that repression is at the level of the chromatin. Indeed, both the MMP9 and MMP13 genes are inaccessible to transcription factors and RNA polymerase II, in association with impaired histone acetylation in their promoters. In accordance with impaired histone acetylation at the cellular level, histone deacetylase-4 is accumulated during HSC transdifferentiation. Furthermore, ectopic expression of histone deacetylase-4 in quiescent HSCs results in repression of MMP promoter activities as well as endogenous MMP9 protein expression. Thus, our findings suggest that a histone deacetylase-4-dependent mechanism underlies the epigenetic silencing of MMP genes during tissue fibrogenesis. PMID:20847282

Induction of miR-137 by isorhapontigenin (ISO) direct targeted Sp1 protein translation and mediated its anti-cancer activity both in vitro and in vivo

PubMed Central

Zeng, Xingruo; Xu, Zhou; Gu, Jiayan; Huang, Haishan; Gao, Guangxun; Zhang, Xiaoru; Li, Jingxia; Jin, Honglei; Jiang, Guosong; Sun, Hong; Huang, Chuanshu

2016-01-01

Our recent studies found that isorhapontigenin (ISO) showed a significant inhibitory effect on human bladder cancer cell growth, accompanied with cell cycle G0/G1 arrest as well as down-regulation of Cyclin D1 expression at transcriptional level via inhibition of Sp1 transactivation in bladder cancer cells. In current studies, the potential ISO inhibition of bladder tumor formation has been explored in xenograft nude mouse model, and the molecular mechanisms underlying ISO inhibition of Sp1 expression and anti-cancer activities has been elucidated both in vitro and in vivo. Moreover, the studies demonstrated that ISO treatment induced the expression of miR-137, which in turn suppressed Sp1 protein translation by direct targeting Sp1 mRNA 3′UTR. Similar to ISO treatment, ectopic expression of miR-137 alone led to G0/G1 cell growth arrest and inhibition of anchorage-independent growth in human bladder cancer cells, which could be completely reversed by over-expression of GFP-Sp1. The inhibition of miR-137 expression attenuated ISO-induced the inhibition of Sp1/Cyclin D1 expression, and induction of G0/G1 cell growth arrest and suppression of cell anchorage-independent growth. Taken together, our studies have demonstrated that miR-137 induction by ISO targets Sp1 mRNA 3′UTR and inhibits Sp1 protein translation, which consequently results in reduction of Cyclin D1 expression, induction of G0/G1 growth arrest and inhibition of anchorage-independent growth in vitro and in vivo. Our results have provided novel insights into understanding the anti-cancer activity of ISO in the therapy of human bladder cancer. PMID:26832795

Cross-regulatory protein-protein interactions between Hox and Pax transcription factors.

PubMed

Plaza, Serge; Prince, Frederic; Adachi, Yoshitsugu; Punzo, Claudio; Cribbs, David L; Gehring, Walter J

2008-09-09

Homeotic Hox selector genes encode highly conserved transcriptional regulators involved in the differentiation of multicellular organisms. Ectopic expression of the Antennapedia (ANTP) homeodomain protein in Drosophila imaginal discs induces distinct phenotypes, including an antenna-to-leg transformation and eye reduction. We have proposed that the eye loss phenotype is a consequence of a negative posttranslational control mechanism because of direct protein-protein interactions between ANTP and Eyeless (EY). In the present work, we analyzed the effect of various ANTP homeodomain mutations for their interaction with EY and for head development. Contrasting with the eye loss phenotype, we provide evidence that the antenna-to-leg transformation involves ANTP DNA-binding activity. In a complementary genetic screen performed in yeast, we isolated mutations located in the N terminus of the ANTP homeodomain that inhibit direct interactions with EY without abolishing DNA binding in vitro and in vivo. In a bimolecular fluorescence complementation assay, we detected the ANTP-EY interaction in vivo, these interactions occurring through the paired domain and/or the homeodomain of EY. These results demonstrate that the homeodomain supports multiple molecular regulatory functions in addition to protein-DNA and protein-RNA interactions; it is also involved in protein-protein interactions.

The QQS orphan gene of Arabidopsis modulates carbon and nitrogen allocation in soybean

PubMed Central

Li, Ling; Wurtele, Eve Syrkin

2015-01-01

The genome of each species contains as high as 8% of genes that are uniquely present in that species. Little is known about the functional significance of these so-called species specific or orphan genes. The Arabidopsis thaliana gene Qua-Quine Starch (QQS) is species specific. Here, we show that altering QQS expression in Arabidopsis affects carbon partitioning to both starch and protein. We hypothesized QQS may be conserved in a feature other than primary sequence, and as such could function to impact composition in another species. To test the potential of QQS in affecting composition in an ectopic species, we introduced QQS into soybean. Soybean T1 lines expressing QQS have up to 80% decreased leaf starch and up to 60% increased leaf protein; T4 generation seeds from field-grown plants contain up to 13% less oil, while protein is increased by up to 18%. These data broaden the concept of QQS as a modulator of carbon and nitrogen allocation, and demonstrate that this species-specific gene can affect the seed composition of an agronomic species thought to have diverged from Arabidopsis 100 million years ago. PMID:25146936

A multicolor panel of novel lentiviral "gene ontology" (LeGO) vectors for functional gene analysis.

PubMed

Weber, Kristoffer; Bartsch, Udo; Stocking, Carol; Fehse, Boris

2008-04-01

Functional gene analysis requires the possibility of overexpression, as well as downregulation of one, or ideally several, potentially interacting genes. Lentiviral vectors are well suited for this purpose as they ensure stable expression of complementary DNAs (cDNAs), as well as short-hairpin RNAs (shRNAs), and can efficiently transduce a wide spectrum of cell targets when packaged within the coat proteins of other viruses. Here we introduce a multicolor panel of novel lentiviral "gene ontology" (LeGO) vectors designed according to the "building blocks" principle. Using a wide spectrum of different fluorescent markers, including drug-selectable enhanced green fluorescent protein (eGFP)- and dTomato-blasticidin-S resistance fusion proteins, LeGO vectors allow simultaneous analysis of multiple genes and shRNAs of interest within single, easily identifiable cells. Furthermore, each functional module is flanked by unique cloning sites, ensuring flexibility and individual optimization. The efficacy of these vectors for analyzing multiple genes in a single cell was demonstrated in several different cell types, including hematopoietic, endothelial, and neural stem and progenitor cells, as well as hepatocytes. LeGO vectors thus represent a valuable tool for investigating gene networks using conditional ectopic expression and knock-down approaches simultaneously.

A Multistate Toggle Switch Defines Fungal Cell Fates and Is Regulated by Synergistic Genetic Cues

PubMed Central

Anderson, Matthew Z.; Porman, Allison M.; Wang, Na; Mancera, Eugenio; Bennett, Richard J.

2016-01-01

Heritable epigenetic changes underlie the ability of cells to differentiate into distinct cell types. Here, we demonstrate that the fungal pathogen Candida tropicalis exhibits multipotency, undergoing stochastic and reversible switching between three cellular states. The three cell states exhibit unique cellular morphologies, growth rates, and global gene expression profiles. Genetic analysis identified six transcription factors that play key roles in regulating cell differentiation. In particular, we show that forced expression of Wor1 or Efg1 transcription factors can be used to manipulate transitions between all three cell states. A model for tristability is proposed in which Wor1 and Efg1 are self-activating but mutually antagonistic transcription factors, thereby forming a symmetrical self-activating toggle switch. We explicitly test this model and show that ectopic expression of WOR1 can induce white-to-hybrid-to-opaque switching, whereas ectopic expression of EFG1 drives switching in the opposite direction, from opaque-to-hybrid-to-white cell states. We also address the stability of induced cell states and demonstrate that stable differentiation events require ectopic gene expression in combination with chromatin-based cues. These studies therefore experimentally test a model of multistate stability and demonstrate that transcriptional circuits act synergistically with chromatin-based changes to drive cell state transitions. We also establish close mechanistic parallels between phenotypic switching in unicellular fungi and cell fate decisions during stem cell reprogramming. PMID:27711197

Spatial distribution of osteoblast-specific transcription factor Cbfa1 and bone formation in atherosclerotic arteries.

PubMed

Bobryshev, Yuri V; Killingsworth, Murray C; Lord, Reginald S A

2008-08-01

The mechanisms of ectopic bone formation in arteries are poorly understood. Osteoblasts might originate either from stem cells that penetrate atherosclerotic plaques from the blood stream or from pluripotent mesenchymal cells that have remained in the arterial wall from embryonic stages of the development. We have examined the frequency of the expression and spatial distribution of osteoblast-specific factor-2/core binding factor-1 (Osf2/Cbfa1) in carotid and coronary arteries. Cbfa1-expressing cells were rarely observed but were found in all tissue specimens in the deep portions of atherosclerotic plaques under the necrotic cores. The deep portions of atherosclerotic plaques under the necrotic cores were characterized by the lack of capillaries of neovascularization. In contrast, plaque shoulders, which were enriched by plexuses of neovascularization, lacked Cbfa1-expressing cells. No bone formation was found in any of the 21 carotid plaques examined and ectopic bone was observed in only two of 12 coronary plaques. We speculate that the sparse invasion of sprouts of neovascularization into areas underlying the necrotic cores, where Cbfa1-expressing cells reside, might explain the rarity of events of ectopic bone formation in the arterial wall. This study has also revealed that Cbfa1-expressing cells contain alpha-smooth muscle actin and myofilaments, indicating their relationship with arterial smooth muscle cells.

Ectopic expression of class 1 KNOX genes induce adventitious shoot regeneration and alter growth and development of tobacco (Nicotiana tabacum L) and European plum (Prunus domestica L).

PubMed

Srinivasan, C; Liu, Zongrang; Scorza, Ralph

2011-04-01

Transgenic plants of tobacco (Nicotiana tabacum L) and European plum (Prunus domestica L) were produced by transforming with the apple class 1 KNOX genes (MdKN1 and MdKN2) or corn KNOX1 gene. Transgenic tobacco plants were regenerated in vitro from transformed leaf discs cultured in a medium lacking cytokinin. Ectopic expression of KNOX genes retarded shoot growth by suppressing elongation of internodes in transgenic tobacco plants. Expression of each of the three KNOX1 genes induced malformation and extensive lobbing in tobacco leaves. In situ regeneration of adventitious shoots was observed from leaves and roots of transgenic tobacco plants expressing each of the three KNOX genes. In vitro culture of leaf explants and internode sections excised from in vitro grown MdKN1 expressing tobacco shoots regenerated adventitious shoots on MS (Murashige and Skoog 1962) basal medium in the absence of exogenous cytokinin. Transgenic plum plants that expressed the MdKN2 or corn KNOX1 gene grew normally but MdKN1 caused a significant reduction in plant height, leaf shape and size and produced malformed curly leaves. A high frequency of adventitious shoot regeneration (96%) was observed in cultures of leaf explants excised from corn KNOX1-expressing transgenic plum shoots. In contrast to KNOX1-expressing tobacco, leaf and internode explants of corn KNOX1-expressing plum required synthetic cytokinin (thidiazuron) in the culture medium to induce adventitious shoot regeneration. The induction of high-frequency regeneration of adventitious shoots in vitro from leaves and stem internodal sections of plum through the ectopic expression of a KNOX1 gene is the first such report for a woody perennial fruit trees.

MicroRNA-205 targets SMAD4 in non-small cell lung cancer and promotes lung cancer cell growth in vitro and in vivo.

PubMed

Zeng, Yuanyuan; Zhu, Jianjie; Shen, Dan; Qin, Hualong; Lei, Zhe; Li, Wei; Liu, Zeyi; Huang, Jian-An

2017-05-09

Despite advances in diagnosis and treatment, the survival of non-small cell lung cancer (NSCLC) patients remains poor; therefore, improved understanding of the disease mechanism and novel treatment strategies are needed. Downregulation of SMAD4 and dysregulated expression of miR-205 have been reported. However, the relationship between them remains unclear. We investigated the effect of microRNA (miR)-205 on the expression of SMAD4 in NSCLC. Knockdown and overexpression of SMAD4 promoted or suppressed cellular viability and proliferation, and accelerated or inhibited the cell cycle in NSCLC cells, respectively. The 3'-untranslated region (3'-UTR) of SMAD4 was predicted as a target of miR-205. Luciferase assays validated that miR-205 binds directly to the SMAD4 3'-UTR. Protein and mRNA expression analyses confirmed that miR-205 overexpression in NSCLC cells inhibited the expression of SMAD4 mRNA and protein. In human NSCLC tissues, increased miR-205 expression was observed frequently and was inversely correlated with decreased SMAD4 expression. Ectopic expression of miR-205 in NSCLC cells suppressed cellular viability and proliferation, accelerated the cell cycle, and promoted tumor growth of lung carcinoma xenografts in nude mice. Our study showed that miR-205 decreased SMAD4 expression, thus promoting NSCLC cell growth. Our findings highlighted the therapeutic potential of targeting miR-205 in NSCLC treatment.

MicroRNA-205 targets SMAD4 in non-small cell lung cancer and promotes lung cancer cell growth in vitro and in vivo

PubMed Central

Qin, Hualong; Lei, Zhe; Li, Wei; Liu, Zeyi; Huang, Jian-an

2017-01-01

Despite advances in diagnosis and treatment, the survival of non-small cell lung cancer (NSCLC) patients remains poor; therefore, improved understanding of the disease mechanism and novel treatment strategies are needed. Downregulation of SMAD4 and dysregulated expression of miR-205 have been reported. However, the relationship between them remains unclear. We investigated the effect of microRNA (miR)-205 on the expression of SMAD4 in NSCLC. Knockdown and overexpression of SMAD4 promoted or suppressed cellular viability and proliferation, and accelerated or inhibited the cell cycle in NSCLC cells, respectively. The 3′-untranslated region (3′-UTR) of SMAD4 was predicted as a target of miR-205. Luciferase assays validated that miR-205 binds directly to the SMAD4 3′-UTR. Protein and mRNA expression analyses confirmed that miR-205 overexpression in NSCLC cells inhibited the expression of SMAD4 mRNA and protein. In human NSCLC tissues, increased miR-205 expression was observed frequently and was inversely correlated with decreased SMAD4 expression. Ectopic expression of miR-205 in NSCLC cells suppressed cellular viability and proliferation, accelerated the cell cycle, and promoted tumor growth of lung carcinoma xenografts in nude mice. Our study showed that miR-205 decreased SMAD4 expression, thus promoting NSCLC cell growth. Our findings highlighted the therapeutic potential of targeting miR-205 in NSCLC treatment. PMID:28199217

Knockdown of p62/sequestosome 1 attenuates autophagy and inhibits colorectal cancer cell growth.

PubMed

Ren, Feng; Shu, Guoshun; Liu, Ganglei; Liu, Dongcai; Zhou, Jiapeng; Yuan, Lianwen; Zhou, Jianping

2014-01-01

p62/sequestosome-1 is a multifunctional adapter protein implicated in selective autophagy, cell signaling pathways, and tumorigenesis, and plays an important role at the crossroad between autophagy and cancer. But, the connection between autophagy and cancer is complex and in some cases contradictory. Human colorectal cancer tissues from patients were analyzed for expression of p62 and Microtubule-associated protein light chain 3 (LC3, an autophagosome marker) using immunostaining, western blotting, real-time PCR, and confocal microscopy. To study the effects of p62 on autophagy and cell growth, shRNA for p62 was applied and cell growth curve was monitored in human colorectal cancer cell. In vivo experiments were done using the mouse xenograft model. We showed that up-regulated expression of p62 and LC3 in colorectal cancer tissues. We also demonstrated that specifically knockdown the expression of p62 showed significantly inhibitory effects not only on autophagy activation, but also on tumor growth both in vitro and xenograft tumors model. The ectopic overexpression of p62 and autophagy activation contributes to colorectal tumorigenesis. p62 and autophagy will be therapy targets for the treatment of colorectal cancer.

A knock-in mouse line conditionally expressing the tumor suppressor WTX/AMER1.

PubMed

Boutet, Agnès; Comai, Glenda; Charlet, Aurélie; Jian Motamedi, Fariba; Dhib, Haroun; Bandiera, Roberto; Schedl, Andreas

2017-11-01

WTX/AMER1 is an important developmental regulator, mutations in which have been identified in a proportion of patients suffering from the renal neoplasm Wilms' tumor and in the bone malformation syndrome Osteopathia Striata with Cranial Sclerosis (OSCS). Its cellular functions appear complex and the protein can be found at the membrane, within the cytoplasm and the nucleus. To understand its developmental and cellular function an allelic series for Wtx in the mouse is crucial. Whereas mice carrying a conditional knock out allele for Wtx have been previously reported, a gain-of-function mouse model that would allow studying the molecular, cellular and developmental role of Wtx is still missing. Here we describe the generation of a novel mouse strain that permits the conditional activation of WTX expression. Wtx fused to GFP was introduced downstream a stop cassette flanked by loxP sites into the Rosa26 locus by gene targeting. Ectopic WTX expression is reported after crosses with several Cre transgenic mice in different embryonic tissues. Further, functionality of the fusion protein was demonstrated in the context of a Wtx null allele. © 2017 Wiley Periodicals, Inc.

Swine interferon-induced transmembrane protein, sIFITM3, inhibits foot-and-mouth disease virus infection in vitro and in vivo.

PubMed

Xu, Jinfang; Qian, Ping; Wu, Qunfeng; Liu, Shasha; Fan, Wenchun; Zhang, Keshan; Wang, Rong; Zhang, Huawei; Chen, Huanchun; Li, Xiangmin

2014-09-01

The interferon-induced transmembrane protein 3 (IFITM3) is a widely expressed potent antiviral effector of the host innate immune system. It restricts a diverse group of pathogenic, enveloped viruses, by interfering with endosomal fusion. In this report, the swine IFITM3 (sIFITM3) gene was cloned. It shares the functionally conserved CD225 domain and multiple critical amino acid residues (Y19, F74, F77, R86 and Y98) with its human ortholog, which are essential for antiviral activity. Ectopic expression of sIFITM3 significantly inhibited non-enveloped foot-and-mouth disease virus (FMDV) infection in BHK-21 cells. Furthermore, sIFITM3 blocked FMDV infection at early steps in the virus life cycle by disrupting viral attachment to the host cell surface. Importantly, inoculation of 2-day-old suckling mice with a plasmid expressing sIFITM3 conferred protection against lethal challenge with FMDV. These results suggest that sIFITM3 is a promising antiviral agent and that can safeguard the host from infection with FMDV. Copyright © 2014 Elsevier B.V. All rights reserved.

Tumor suppressor activity of the ERK/MAPK pathway by promoting selective protein degradation

PubMed Central

Deschênes-Simard, Xavier; Gaumont-Leclerc, Marie-France; Bourdeau, Véronique; Lessard, Frédéric; Moiseeva, Olga; Forest, Valérie; Igelmann, Sebastian; Mallette, Frédérick A.; Saba-El-Leil, Marc K.; Meloche, Sylvain; Saad, Fred; Mes-Masson, Anne-Marie; Ferbeyre, Gerardo

2013-01-01

Constitutive activation of growth factor signaling pathways paradoxically triggers a cell cycle arrest known as cellular senescence. In primary cells expressing oncogenic ras, this mechanism effectively prevents cell transformation. Surprisingly, attenuation of ERK/MAP kinase signaling by genetic inactivation of Erk2, RNAi-mediated knockdown of ERK1 or ERK2, or MEK inhibitors prevented the activation of the senescence mechanism, allowing oncogenic ras to transform primary cells. Mechanistically, ERK-mediated senescence involved the proteasome-dependent degradation of proteins required for cell cycle progression, mitochondrial functions, cell migration, RNA metabolism, and cell signaling. This senescence-associated protein degradation (SAPD) was observed not only in cells expressing ectopic ras, but also in cells that senesced due to short telomeres. Individual RNAi-mediated inactivation of SAPD targets was sufficient to restore senescence in cells transformed by oncogenic ras or trigger senescence in normal cells. Conversely, the anti-senescence viral oncoproteins E1A, E6, and E7 prevented SAPD. In human prostate neoplasms, high levels of phosphorylated ERK were found in benign lesions, correlating with other senescence markers and low levels of STAT3, one of the SAPD targets. We thus identified a mechanism that links aberrant activation of growth signaling pathways and short telomeres to protein degradation and cellular senescence. PMID:23599344

Osteoprotegerin Promotes Cementoblastic Activity of Murine Cementoblast Cell Line in vitro.

PubMed

Zhang, Ying Ying; Zhao, Hua Xiang; Chen, Zhi Bin; Lin, Jiu Xiang; Liu, Yan

2016-06-01

To investigate the effect of osteoprotegerin (OPG) on the cementoblastic activity of a clonal population of immortalised murine cementoblasts (OCCM-30) in vitro. OCCM-30 cells were transiently transfected with the mouse OPG using the Avalanche transfection reagent. The ectopic expression of OPG was confirmed by enzyme-linked immunosorbent assay and quantitative polymerase chain reaction. The cell counting Kit-8 assay was used to investigate the effect of OPG on cell proliferation. The expression levels of cementoblastic-related mRNA and protein in the transfected OCCM-30 cells were detected using real-time PCR, Western blotting and immunohistochemical staining. Satisfactory transfection efficiency was observed 48 h after transfection. The results of the cell proliferation assay indicated that the expansion rate of the OPG transfection group was greater than that of the control group at both 72 h and 96 h. The mRNA levels of osterix (Osx), protein kinase B (Akt1), cementum attachment protein (CAP) and osteopontin (Opn) were significantly upregulated (P < 0.05) in the OPG group. Protein levels of OPN, bone sialoprotein II (BSP II), osteocalcin (OC) and CAP, which are responsible for osteogenetic and cementoblastic activity, were significantly increased in the OPG-overexpressing group. Overexpression of OPG in OCCM-30 cells promotes cementoblastic activity.

Distinct Contributions of Conserved Modules to Runt Transcription Factor Activity

PubMed Central

Walrad, Pegine B.; Hang, Saiyu; Joseph, Genevieve S.; Salas, Julia

2010-01-01

Runx proteins play vital roles in regulating transcription in numerous developmental pathways throughout the animal kingdom. Two Runx protein hallmarks are the DNA-binding Runt domain and a C-terminal VWRPY motif that mediates interaction with TLE/Gro corepressor proteins. A phylogenetic analysis of Runt, the founding Runx family member, identifies four distinct regions C-terminal to the Runt domain that are conserved in Drosophila and other insects. We used a series of previously described ectopic expression assays to investigate the functions of these different conserved regions in regulating gene expression during embryogenesis and in controlling axonal projections in the developing eye. The results indicate each conserved region is required for a different subset of activities and identify distinct regions that participate in the transcriptional activation and repression of the segmentation gene sloppy-paired-1 (slp1). Interestingly, the C-terminal VWRPY-containing region is not required for repression but instead plays a role in slp1 activation. Genetic experiments indicating that Groucho (Gro) does not participate in slp1 regulation further suggest that Runt's conserved C-terminus interacts with other factors to promote transcriptional activation. These results provide a foundation for further studies on the molecular interactions that contribute to the context-dependent properties of Runx proteins as developmental regulators. PMID:20462957

A high affinity RIM-binding protein/Aplip1 interaction prevents the formation of ectopic axonal active zones.

PubMed

Siebert, Matthias; Böhme, Mathias A; Driller, Jan H; Babikir, Husam; Mampell, Malou M; Rey, Ulises; Ramesh, Niraja; Matkovic, Tanja; Holton, Nicole; Reddy-Alla, Suneel; Göttfert, Fabian; Kamin, Dirk; Quentin, Christine; Klinedinst, Susan; Andlauer, Till Fm; Hell, Stefan W; Collins, Catherine A; Wahl, Markus C; Loll, Bernhard; Sigrist, Stephan J

2015-08-14

Synaptic vesicles (SVs) fuse at active zones (AZs) covered by a protein scaffold, at Drosophila synapses comprised of ELKS family member Bruchpilot (BRP) and RIM-binding protein (RBP). We here demonstrate axonal co-transport of BRP and RBP using intravital live imaging, with both proteins co-accumulating in axonal aggregates of several transport mutants. RBP, via its C-terminal Src-homology 3 (SH3) domains, binds Aplip1/JIP1, a transport adaptor involved in kinesin-dependent SV transport. We show in atomic detail that RBP C-terminal SH3 domains bind a proline-rich (PxxP) motif of Aplip1/JIP1 with submicromolar affinity. Pointmutating this PxxP motif provoked formation of ectopic AZ-like structures at axonal membranes. Direct interactions between AZ proteins and transport adaptors seem to provide complex avidity and shield synaptic interaction surfaces of pre-assembled scaffold protein transport complexes, thus, favouring physiological synaptic AZ assembly over premature assembly at axonal membranes.

Alteration of flower color in Iris germanica L. 'Fire Bride' through ectopic expression of phytoene synthase gene (crtB) from Pantoea agglomerans.

PubMed

Jeknić, Zoran; Jeknić, Stevan; Jevremović, Slađana; Subotić, Angelina; Chen, Tony H H

2014-08-01

Genetic modulation of the carotenogenesis in I. germanica 'Fire Bride' by ectopic expression of a crtB gene causes several flower parts to develop novel orange and pink colors. Flower color in tall bearded irises (Iris germanica L.) is determined by two distinct biochemical pathways; the carotenoid pathway, which imparts yellow, orange and pink hues and the anthocyanin pathway, which produces blue, violet and maroon flowers. Red-flowered I. germanica do not exist in nature and conventional breeding methods have thus far failed to produce them. With a goal of developing iris cultivars with red flowers, we transformed a pink iris I. germanica, 'Fire Bride', with a bacterial phytoene synthase gene (crtB) from Pantoea agglomerans under the control of the promoter region of a gene for capsanthin-capsorubin synthase from Lilium lancifolium (Llccs). This approach aimed to increase the flux of metabolites into the carotenoid biosynthetic pathway and lead to elevated levels of lycopene and darker pink or red flowers. Iris callus tissue ectopically expressing the crtB gene exhibited a color change from yellow to pink-orange and red, due to accumulation of lycopene. Transgenic iris plants, regenerated from the crtB-transgenic calli, showed prominent color changes in the ovaries (green to orange), flower stalk (green to orange), and anthers (white to pink), while the standards and falls showed no significant differences in color when compared to control plants. HPLC and UHPLC analysis confirmed that the color changes were primarily due to the accumulation of lycopene. In this study, we showed that ectopic expression of a crtB can be used to successfully alter the color of certain flower parts in I. germanica 'Fire Bride' and produce new flower traits.

Failure of in vitro-differentiated mesenchymal stem cells from the synovial membrane to form ectopic stable cartilage in vivo.

PubMed

De Bari, Cosimo; Dell'Accio, Francesco; Luyten, Frank P

2004-01-01

We previously reported the identification in a nude mouse assay of molecular markers predictive of the capacity of articular cartilage-derived cells (ACDCs) to form ectopic stable cartilage that is resistant to vascular invasion and endochondral ossification. In the present study, we investigated whether in vitro-differentiated mesenchymal stem cells (MSCs) from the synovial membrane (SM) express the stable-chondrocyte markers and form ectopic stable cartilage in vivo. Chondrogenesis was induced in micromass culture with the addition of transforming growth factor beta1 (TGFbeta1). After acquisition of the cartilage phenotype, micromasses were implanted subcutaneously into nude mice. Alternatively, cells were released enzymatically and either replated in monolayer or injected intramuscularly into nude mice. Marker analysis was performed by quantitative reverse transcription-polymerase chain reaction. Cell death was detected with TUNEL assay. Cartilage-like micromasses and released cells expressed the stable-chondrocyte markers at levels comparable with those expressed by stable ACDCs. The released cells lost chondrocyte marker expression by 24 hours in monolayer and failed to form cartilage when injected intramuscularly into nude mice. Instead, myogenic differentiation was detected. When intact TGFbeta1-treated micromasses were implanted subcutaneously, they partially lost their cartilage phenotype and underwent cell death and neoangiogenesis within 1 week. At later time points (15-40 days), we retrieved neither cartilage nor bone, and human cells were not detectable. The chondrocyte-like phenotype of human SM MSCs, induced in vitro under specific conditions, appears to be unstable and is not sufficient to obtain ectopic formation of stable cartilage in vivo. Studies in animal models of joint surface defect repair are necessary to evaluate the stability of the SM MSC chondrocyte-like phenotype within the joint environment.

Mechanisms of protein kinase C signaling in the modulation of 3',5'-cyclic adenosine monophosphate-mediated steroidogenesis in mouse gonadal cells.

PubMed

Manna, Pulak R; Huhtaniemi, Ilpo T; Stocco, Douglas M

2009-07-01

The protein kinase C (PKC) signaling pathway plays integral roles in the expression of the steroidogenic acute regulatory (StAR) protein that regulates steroid biosynthesis in steroidogenic cells. PKC can modulate the activity of cAMP/protein kinase A signaling involved in steroidogenesis; however, its mechanism remains obscure. In the present study, we demonstrate that activation of the PKC pathway, by phorbol 12-myristate 13-acetate (PMA), was capable of potentiating dibutyryl cAMP [(Bu)(2)cAMP]-stimulated StAR expression, StAR phosphorylation, and progesterone synthesis in both mouse Leydig (MA-10) and granulosa (KK-1) tumor cells. The steroidogenic potential of PMA and (Bu)(2)cAMP was linked with phosphorylation of ERK 1/2; however, inhibition of the latter demonstrated varying effects on steroidogenesis. Transcriptional activation of the StAR gene by PMA and (Bu)(2)cAMP was influenced by several factors, its up-regulation being dependent on phosphorylation of the cAMP response element binding protein (CREB). An oligonucleotide probe containing a CREB/activating transcription factor binding region in the StAR promoter was found to bind nuclear proteins in PMA and (Bu)(2)cAMP-treated MA-10 and KK-1 cells. Chromatin immunoprecipitation studies revealed that the induction of phosphorylated CREB was tightly correlated with in vivo protein-DNA interactions and recruitment of CREB binding protein to the StAR promoter. Ectopic expression of CREB binding protein enhanced CREB-mediated transcription of the StAR gene, an event that was markedly repressed by the adenovirus E1A oncoprotein. Further studies demonstrated that the activation of StAR expression and steroid synthesis by PMA and (Bu)(2)cAMP was associated with expression of the nuclear receptor Nur77, indicating its essential role in hormone-regulated steroidogenesis. Collectively, these findings provide insight into the mechanisms by which PKC modulates cAMP/protein kinase A responsiveness involved in regulating the steroidogenic response in mouse gonadal cells.

Ectopic overexpression of LAPTM5 results in lysosomal targeting and induces Mcl-1 down-regulation, Bak activation, and mitochondria-dependent apoptosis in human HeLa cells

PubMed Central

Jun, Do Youn; Kim, Hyejin; Jang, Won Young; Lee, Ji Young; Fukui, Kiyoshi; Kim, Young Ho

2017-01-01

Human lysosomal-associated protein multispanning membrane 5 (LAPTM5) was identified by an ordered differential display-polymerase chain reaction (ODD-PCR) as an up-regulated cDNA fragment during 12-O-tetradecanoylphorbol 13-acetate (TPA)-induced differentiation of U937 cells into monocytes/macrophages. After TPA-treatment, the levels of LAPTM5 mRNA and protein increased and reached a maximum at 18–36 h. In healthy human tissues, LAPTM5 mRNA was expressed at high levels in hematopoietic cells and tissues, at low levels in the lung and fetal liver, and was not detected in other non-hematopoietic tissues. LAPTM5 mRNA was detected in immature malignant cells of myeloid lineage, such as K562, HL-60, U937, and THP-1 cells, and in unstimulated peripheral T cells, but was absent or barely detectable in lymphoid malignant or non-hematopoietic malignant cells. The LAPTM5 level in HL-60 cells increased more significantly during TPA-induced monocyte/macrophage differentiation than during DMSO-induced granulocyte differentiation. Ectopic expression of GFP-LAPTM5 or LAPTM5 in HeLa cells exhibited the localization of LAPTM5 to the lysosome. In HeLa cells overexpressing LAPTM5, the Mcl-1 and Bid levels declined markedly and apoptosis was induced via Bak activation, Δψm loss, activation of caspase-9, -8 and -3, and PARP degradation without accompanying necrosis. However, these LAPTM5-induced apoptotic events except for the decline of Bid level were completely abrogated by concomitant overexpression of Mcl-1. The pan-caspase inhibitor (z-VAD-fmk) could suppress the LAPTM5-induced apoptotic sub-G1 peak by ~40% but failed to block the induced Δψm loss, whereas the broad-range inhibitor of cathepsins (Cathepsin Inhibitor I) could suppress the LAPTM5-induced apoptotic sub-G1 peak and Δψm loss, by ~22% and ~23%, respectively, suggesting that the LAPTM5-mediated Δψm loss was exerted at least in part in a cathepsin-dependent manner. Together, these results demonstrate that ectopic overexpression of LAPTM5 in HeLa cells induced apoptosis via cleavage of Mcl-1 and Bid by a LAPTM5-associated lysosomal pathway, and subsequent activation of the mitochondria-dependent caspase cascade. PMID:28464033

Hepatitis C Virus Core Protein Modulates Endoglin (CD105) Signaling Pathway for Liver Pathogenesis.

PubMed

Kwon, Young-Chan; Sasaki, Reina; Meyer, Keith; Ray, Ranjit

2017-11-01

Endoglin is part of the TGF-β receptor complex and has a crucial role in fibrogenesis and angiogenesis. It is also an important protein for tumor growth, survival, and cancer cell metastasis. In a previous study, we have shown that hepatitis C virus (HCV) infection induces epithelial-mesenchymal transition (EMT) state and cancer stem-like cell (CSC) properties in human hepatocytes. Our array data suggested that endoglin (CD105) mRNA is significantly upregulated in HCV-associated CSCs. In this study, we have observed increased endoglin expression on the cell surface of an HCV core-expressing hepatocellular carcinoma (HepG2) cell line or immortalized human hepatocytes (IHH) and activation of its downstream signaling molecules. The status of phospho-SMAD1/5 and the expression of inhibitor of DNA binding protein 1 (ID1) were upregulated in HCV-infected cells or viral core gene-transfected cells. Additionally, we observed upregulation of endoglin/ID1 mRNA expression in chronic HCV patient liver biopsy samples. CSC generation by HCV core protein was dependent on the endoglin signaling pathway using activin receptor-like kinase 1 (ALK1) Fc blocking peptide and endoglin small interfering RNA (siRNA). Further, follow-up from in vitro analysis suggested that the antiapoptosis Bcl2 protein, proliferation-related cyclin D1 protein, and CSC-associated Hes1, Notch1, Nanog, and Sox2 proteins are enhanced during infection or ectopic expression of HCV core protein. IMPORTANCE Endoglin plays a crucial role in fibrogenesis and angiogenesis and is an important protein for tumor growth, survival, and cancer cell metastasis. Endoglin enhances ALK1-SMAD1/5 signaling in different cell types, leading to increased proliferation and migration responses. We have observed endoglin expression on the HCV core-expressing cell surface of human hepatocyte origin and activation of phospho-SMAD1/5 and ID1 downstream signaling molecules. ID1 protein plays a role in CSC properties, and we found that this pathway is important for antiapoptotic and cell proliferation signaling. Blocking of endoglin-ALK1-SMAD1/5 might be a good candidate for therapy for liver cancer stem cells together with liver cirrhosis. Copyright © 2017 American Society for Microbiology.

Ideal crop plant architecture is mediated by tassels replace upper ears1, a BTB/POZ ankyrin repeat gene directly targeted by TEOSINTE BRANCHED1.

PubMed

Dong, Zhaobin; Li, Wei; Unger-Wallace, Erica; Yang, Jinliang; Vollbrecht, Erik; Chuck, George

2017-10-10

Axillary branch suppression is a favorable trait bred into many domesticated crop plants including maize compared with its highly branched wild ancestor teosinte. Branch suppression in maize was achieved through selection of a gain of function allele of the teosinte branched1 (tb1) transcription factor that acts as a repressor of axillary bud growth. Previous work indicated that other loci may function epistatically with tb1 and may be responsible for some of its phenotypic effects. Here, we show that tb1 mediates axillary branch suppression through direct activation of the tassels replace upper ears1 ( tru1 ) gene that encodes an ankyrin repeat domain protein containing a BTB/POZ motif necessary for protein-protein interactions. The expression of TRU1 and TB1 overlap in axillary buds, and TB1 binds to two locations in the tru1 gene as shown by chromatin immunoprecipitation and gel shifts. In addition, nucleotide diversity surveys indicate that tru1 , like tb1 , was a target of selection. In modern maize, TRU1 is highly expressed in the leaf trace vasculature of axillary internodes, while in teosinte, this expression is highly reduced or absent. This increase in TRU1 expression levels in modern maize is supported by comparisons of relative protein levels with teosinte as well as by quantitative measurements of mRNA levels. Hence, a major innovation in creating ideal maize plant architecture originated from ectopic overexpression of tru1 in axillary branches, a critical step in mediating the effects of domestication by tb1.

Ectopic expression of R3 MYB transcription factor gene OsTCL1 in Arabidopsis, but not rice, affects trichome and root hair formation

PubMed Central

Zheng, Kaijie; Tian, Hainan; Hu, Qingnan; Guo, Hongyan; Yang, Li; Cai, Ling; Wang, Xutong; Liu, Bao; Wang, Shucai

2016-01-01

In Arabidopsis, a MYB-bHLH-WD40 (MBW) transcriptional activator complex activates the homeodomain protein gene GLABRA2 (GL2), leading to the promotion of trichome formation and inhibition of root hair formation. The same MBW complex also activates single-repeat R3 MYB genes. R3 MYBs in turn, play a negative feedback role by competing with R2R3 MYB proteins for binding bHLH proteins, thus blocking the formation of the MBW complex. By BLASTing the rice (Oryza sativa) protein database using the entire amino acid sequence of Arabidopsis R3 MYB transcription factor TRICHOMELESS1 (TCL1), we found that there are two genes in rice genome encoding R3 MYB transcription factors, namely Oryza sativa TRICHOMELESS1 (OsTCL1) and OsTCL2. Expressing OsTCL1 in Arabidopsis inhibited trichome formation and promoted root hair formation, and OsTCL1 interacted with GL3 when tested in Arabidopsis protoplasts. Consistent with these observations, expression levels of GL2, R2R3 MYB transcription factor gene GLABRA1 (GL1) and several R3 MYB genes were greatly reduced, indicating that OsTCL1 is functional R3 MYB. However, trichome and root hair formation in transgenic rice plants overexpressing OsTCL1 remained largely unchanged, and elevated expression of OsGL2 was observed in the transgenic rice plants, indicating that rice may use different mechanisms to regulate trichome formation. PMID:26758286

Foxp3-dependent transformation of human primary CD4+ T lymphocytes by the retroviral protein tax.

PubMed

Chen, Li; Liu, Dan; Zhang, Yang; Zhang, Huan; Cheng, Hua

2015-10-23

The retroviral Tax proteins of human T cell leukemia virus type 1 and 2 (HTLV-1 and -2) are highly homologous viral transactivators. Both viral proteins can immortalize human primary CD4+ memory T cells, but when expressed alone they rarely transform T cells. In the present study, we found that the Tax proteins displayed a differential ability to immortalize human CD4+Foxp3+ T cells with characteristic expression of CTLA-4 and GITR. Because epidermal growth factor receptor (EGFR) was reportedly expressed and activated in a subset of CD4+Foxp3+ T cells, we introduced an activated EGFR into Tax-immortalized CD4+Foxp3+ T cells. We observed that these modified cells were grown independently of exogenous IL-2, correlating with a T cell transformation phenotype. In Tax-immortalized CD4+Foxp3- T cells, ectopic expression of Foxp3 was a prerequisite for Tax transformation of T cells. Accordingly, treatment of the transformed T cells with erlotinib, a selective inhibitor of EGFR, induced degradation of EGFR in lysosome, consequently causing T cell growth inhibition. Further, we identified autophagy as a crucial cellular survival pathway for the transformed T cells. Silencing key autophagy molecules including Beclin1, Atg5 and PI3 kinase class III (PI3KC3) resulted in drastic impairment of T cell growth. Our data, therefore, unveiled a previously unidentified role of Foxp3 in T cell transformation, providing a molecular basis for HTLV-1 transformation of CD4+Foxp3+ T cells. Copyright © 2015 Elsevier Inc. All rights reserved.

Foxp3-dependent Transformation of Human Primary CD4+ T Lymphocytes by the Retroviral Protein Tax

PubMed Central

Chen, Li; Liu, Dan; Zhang, Yang; Zhang, Huan; Cheng, Hua

2015-01-01

The retroviral Tax proteins of human T cell leukemia virus type 1 and 2 (HTLV-1 and -2) are highly homologous viral transactivators. Both viral proteins can immortalize human primary CD4+ memory T cells, but when expressed alone they rarely transform T cells. In the present study, we found that the Tax proteins displayed a differential ability to immortalize human CD4+Foxp3+ T cells with characteristic expression of CTLA-4 and GITR. Because epidermal growth factor receptor (EGFR) was reportedly expressed and activated in a subset of CD4+Foxp3+ T cells, we introduced an activated EGFR into Tax-immortalized CD4+Foxp3+ T cells. We observed that these modified cells were grown independently of exogenous IL-2, correlating with a T cell transformation phenotype. In Tax-immortalized CD4+Foxp3- T cells, ectopic expression of Foxp3 was a prerequisite for Tax transformation of T cells. Accordingly, treatment of the transformed T cells with erlotinib, a selective inhibitor of EGFR, induced degradation of EGFR in lysosome, consequently causing T cell growth inhibition. Further, we identified autophagy as a crucial cellular survival pathway for the transformed T cells. Silencing key autophagy molecules including Beclin1, Atg5 and PI3 kinase class III (PI3KC3) resulted in drastic impairment of T cell growth. Our data, therefore, unveiled a previously unidentified role of Foxp3 in T cell transformation, providing a molecular basis for HTLV-1 transformation of CD4+Foxp3+ T cells. PMID:26381169

SATB2 expression increased anchorage-independent growth and cell migration in human bronchial epithelial cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wu, Feng; Jordan, Ashley; Kluz, Thomas

The special AT-rich sequence-binding protein 2 (SATB2) is a protein that binds to the nuclear matrix attachment region of the cell and regulates gene expression by altering chromatin structure. In our previous study, we reported that SATB2 gene expression was induced in human bronchial epithelial BEAS-2B cells transformed by arsenic, chromium, nickel and vanadium. In this study, we show that ectopic expression of SATB2 in the normal human bronchial epithelial cell-line BEAS-2B increased anchorage-independent growth and cell migration, meanwhile, shRNA-mediated knockdown of SATB2 significantly decreased anchorage-independent growth in Ni transformed BEAS-2B cells. RNA sequencing analyses of SATB2 regulated genes revealedmore » the enrichment of those involved in cytoskeleton, cell adhesion and cell-movement pathways. Our evidence supports the hypothesis that SATB2 plays an important role in BEAS-2B cell transformation. - Highlights: • We performed SATB2 overexpression in the BEAS-2B cell line. • We performed SATB2 knockdown in a Ni transformed BEAS-2B cell line. • SATB2 induced anchorage-independent growth and increased cell migration. • SATB2 knockdown significantly decreased anchorage-independent growth. • We identified alterations in gene involved in cytoskeleton, cell adhesion.« less

SOX10 Regulates Expression of the SH3-Domain Kinase Binding Protein 1 (Sh3kbp1) locus in Schwann Cells via an Alternative Promoter

PubMed Central

Hodonsky, Chani J.; Kleinbrink, Erica L.; Charney, Kira N.; Prasad, Megana; Bessling, Seneca L.; Jones, Erin A.; Srinivasan, Rajini; Svaren, John; McCallion, Andrew S.; Antonellis, Anthony

2011-01-01

The transcription factor SOX10 has essential roles in neural crest-derived cell populations, including myelinating Schwann cells—specialized glial cells responsible for ensheathing axons in the peripheral nervous system. Importantly, SOX10 directly regulates the expression of genes essential for proper myelin function. To date, only a handful of SOX10 target loci have been characterized in Schwann cells. Addressing this lack of knowledge will provide a better understanding of Schwann cell biology and candidate loci for relevant diseases such as demyelinating peripheral neuropathies. We have identified a highly-conserved SOX10 binding site within an alternative promoter at the SH3-domain kinase binding protein 1 (Sh3kbp1) locus. The genomic segment identified at Sh3kbp1 binds to SOX10 and displays strong promoter activity in Schwann cells in vitro and in vivo. Mutation of the SOX10 binding site ablates promoter activity, and ectopic expression of SOX10 in SOX10-negative cells promotes the expression of endogenous Sh3kbp1. Combined, these data reveal Sh3kbp1 as a novel target of SOX10 and raise important questions regarding the function of SH3KBP1 isoforms in Schwann cells. PMID:22037207

Mouse fat storage-inducing transmembrane protein 2 (FIT2) promotes lipid droplet accumulation in plants.

PubMed

Cai, Yingqi; McClinchie, Elizabeth; Price, Ann; Nguyen, Thuy N; Gidda, Satinder K; Watt, Samantha C; Yurchenko, Olga; Park, Sunjung; Sturtevant, Drew; Mullen, Robert T; Dyer, John M; Chapman, Kent D

2017-07-01

Fat storage-inducing transmembrane protein 2 (FIT2) is an endoplasmic reticulum (ER)-localized protein that plays an important role in lipid droplet (LD) formation in animal cells. However, no obvious homologue of FIT2 is found in plants. Here, we tested the function of FIT2 in plant cells by ectopically expressing mouse (Mus musculus) FIT2 in Nicotiana tabacum suspension-cultured cells, Nicotiana benthamiana leaves and Arabidopsis thaliana plants. Confocal microscopy indicated that the expression of FIT2 dramatically increased the number and size of LDs in leaves of N. benthamiana and Arabidopsis, and lipidomics analysis and mass spectrometry imaging confirmed the accumulation of neutral lipids in leaves. FIT2 also increased seed oil content by ~13% in some stable, overexpressing lines of Arabidopsis. When expressed transiently in leaves of N. benthamiana or suspension cells of N. tabacum, FIT2 localized specifically to the ER and was often concentrated at certain regions of the ER that resembled ER-LD junction sites. FIT2 also colocalized at the ER with other proteins known to be involved in triacylglycerol biosynthesis or LD formation in plants, but not with ER resident proteins involved in electron transfer or ER-vesicle exit sites. Collectively, these results demonstrate that mouse FIT2 promotes LD accumulation in plants, a surprising functional conservation in the context of a plant cell given the apparent lack of FIT2 homologues in higher plants. These results suggest also that FIT2 expression represents an effective synthetic biology strategy for elaborating neutral lipid compartments in plant tissues for potential biofuel or bioproduct purposes. © 2016 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

Mouse fat storage-inducing transmembrane protein 2 (FIT2) promotes lipid droplet accumulation in plants

DOE PAGES

Cai, Yingqi; McClinchie, Elizabeth; Price, Ann; ...

2017-01-18

Fat storage-inducing transmembrane protein 2 (FIT2) is an endoplasmic reticulum (ER)-localized protein that plays an important role in lipid droplet (LD) formation in animal cells. However, no obvious homologue of FIT2 is found in plants. We tested the function of FIT2 in plant cells by ectopically expressing mouse (Mus musculus) FIT2 in Nicotiana tabacum suspension-cultured cells, Nicotiana benthamiana leaves and Arabidopsis thaliana plants. Confocal microscopy indicated that the expression of FIT2 dramatically increased the number and size of LDs in leaves of N. benthamiana and Arabidopsis, and lipidomics analysis and mass spectrometry imaging confirmed the accumulation of neutral lipids inmore » leaves. FIT2 also increased seed oil content by ~13% in some stable, overexpressing lines of Arabidopsis. Furthermore, when expressed transiently in leaves of N. benthamiana or suspension cells of N. tabacum, FIT2 localized specifically to the ER and was often concentrated at certain regions of the ER that resembled ER-LD junction sites. FIT2 also colocalized at the ER with other proteins known to be involved in triacylglycerol biosynthesis or LD formation in plants, but not with ER resident proteins involved in electron transfer or ERvesicle exit sites. Collectively, these results demonstrate that mouse FIT2 promotes LD accumulation in plants, a surprising functional conservation in the context of a plant cell given the apparent lack of FIT2 homologues in higher plants. Our results suggest also that FIT2 expression represents an effective synthetic biology strategy for elaborating neutral lipid compartments in plant tissues for potential biofuel or bioproduct purposes.« less

Mouse fat storage-inducing transmembrane protein 2 (FIT2) promotes lipid droplet accumulation in plants

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cai, Yingqi; McClinchie, Elizabeth; Price, Ann

Fat storage-inducing transmembrane protein 2 (FIT2) is an endoplasmic reticulum (ER)-localized protein that plays an important role in lipid droplet (LD) formation in animal cells. However, no obvious homologue of FIT2 is found in plants. We tested the function of FIT2 in plant cells by ectopically expressing mouse (Mus musculus) FIT2 in Nicotiana tabacum suspension-cultured cells, Nicotiana benthamiana leaves and Arabidopsis thaliana plants. Confocal microscopy indicated that the expression of FIT2 dramatically increased the number and size of LDs in leaves of N. benthamiana and Arabidopsis, and lipidomics analysis and mass spectrometry imaging confirmed the accumulation of neutral lipids inmore » leaves. FIT2 also increased seed oil content by ~13% in some stable, overexpressing lines of Arabidopsis. Furthermore, when expressed transiently in leaves of N. benthamiana or suspension cells of N. tabacum, FIT2 localized specifically to the ER and was often concentrated at certain regions of the ER that resembled ER-LD junction sites. FIT2 also colocalized at the ER with other proteins known to be involved in triacylglycerol biosynthesis or LD formation in plants, but not with ER resident proteins involved in electron transfer or ERvesicle exit sites. Collectively, these results demonstrate that mouse FIT2 promotes LD accumulation in plants, a surprising functional conservation in the context of a plant cell given the apparent lack of FIT2 homologues in higher plants. Our results suggest also that FIT2 expression represents an effective synthetic biology strategy for elaborating neutral lipid compartments in plant tissues for potential biofuel or bioproduct purposes.« less

A Novel Post-translational Modification of Nucleolin, SUMOylation at Lys-294, Mediates Arsenite-induced Cell Death by Regulating gadd45α mRNA Stability*

PubMed Central

Zhang, Dongyun; Liang, Yuguang; Xie, Qipeng; Gao, Guangxun; Wei, Jinlong; Huang, Haishan; Li, Jingxia; Gao, Jimin; Huang, Chuanshu

2015-01-01

Nucleolin is a ubiquitously expressed protein and participates in many important biological processes, such as cell cycle regulation and ribosomal biogenesis. The activity of nucleolin is regulated by intracellular localization and post-translational modifications, including phosphorylation, methylation, and ADP-ribosylation. Small ubiquitin-like modifier (SUMO) is a category of recently verified forms of post-translational modifications and exerts various effects on the target proteins. In the studies reported here, we discovered SUMOylational modification of human nucleolin protein at Lys-294, which facilitated the mRNA binding property of nucleolin by maintaining its nuclear localization. In response to arsenic exposure, nucleolin-SUMO was induced and promoted its binding with gadd45α mRNA, which increased gadd45α mRNA stability and protein expression, subsequently causing GADD45α-mediated cell death. On the other hand, ectopic expression of Mn-SOD attenuated the arsenite-generated superoxide radical level, abrogated nucleolin-SUMO, and in turn inhibited arsenite-induced apoptosis by reducing GADD45α expression. Collectively, our results for the first time demonstrate that nucleolin-SUMO at K294R plays a critical role in its nucleus sequestration and gadd45α mRNA binding activity. This novel biological function of nucleolin is distinct from its conventional role as a proto-oncogene. Therefore, our findings here not only reveal a new modification of nucleolin protein and its novel functional paradigm in mRNA metabolism but also expand our understanding of the dichotomous roles of nucleolin in terms of cancer development, which are dependent on multiple intracellular conditions and consequently the appropriate regulations of its modifications, including SUMOylation. PMID:25561743

Ki-1/57 and CGI-55 ectopic expression impact cellular pathways involved in proliferation and stress response regulation.

PubMed

Costa, Fernanda C; Saito, Angela; Gonçalves, Kaliandra A; Vidigal, Pedro M; Meirelles, Gabriela V; Bressan, Gustavo C; Kobarg, Jörg

2014-12-01

Ki-1/57 (HABP4) and CGI-55 (SERBP1) are regulatory proteins and paralogs with 40.7% amino acid sequence identity and 67.4% similarity. Functionally, they have been implicated in the regulation of gene expression on both the transcriptional and mRNA metabolism levels. A link with tumorigenesis is suggested, since both paralogs show altered expression levels in tumor cells and the Ki-1/57 gene is found in a region of chromosome 9q that represents a haplotype for familiar colon cancer. However, the target genes regulated by Ki-1/57 and CGI-55 are unknown. Here, we analyzed the alterations of the global transcriptome profile after Ki-1/57 or CGI-55 overexpression in HEK293T cells by DNA microchip technology. We were able to identify 363 or 190 down-regulated and 50 or 27 up-regulated genes for Ki-1/57 and CGI-55, respectively, of which 20 were shared between both proteins. Expression levels of selected genes were confirmed by qRT-PCR both after protein overexpression and siRNA knockdown. The majority of the genes with altered expression were associated to proliferation, apoptosis and cell cycle control processes, prompting us to further explore these contexts experimentally. We observed that overexpression of Ki-1/57 or CGI-55 results in reduced cell proliferation, mainly due to a G1 phase arrest, whereas siRNA knockdown of CGI-55 caused an increase in proliferation. In the case of Ki-1/57 overexpression, we found protection from apoptosis after treatment with the ER-stress inducer thapsigargin. Together, our data give important new insights that may help to explain these proteins putative involvement in tumorigenic events. Copyright © 2014 Elsevier B.V. All rights reserved.

Flotillin proteins recruit sphingosine to membranes and maintain cellular sphingosine-1-phosphate levels

PubMed Central

Riento, Kirsi; Zhang, Qifeng; Clark, Jonathan; Begum, Farida; Stephens, Elaine; Wakelam, Michael J.

2018-01-01

Sphingosine-1-phosphate (S1P) is an important lipid signalling molecule. S1P is produced via intracellular phosphorylation of sphingosine (Sph). As a lipid with a single fatty alkyl chain, Sph may diffuse rapidly between cellular membranes and through the aqueous phase. Here, we show that the absence of microdomains generated by multimeric assemblies of flotillin proteins results in reduced S1P levels. Cellular phenotypes of flotillin knockout mice, including changes in histone acetylation and expression of Isg15, are recapitulated when S1P synthesis is perturbed. Flotillins bind to Sph in vitro and increase recruitment of Sph to membranes in cells. Ectopic re-localisation of flotillins within the cell causes concomitant redistribution of Sph. The data suggest that flotillins may directly or indirectly regulate cellular sphingolipid distribution and signalling. PMID:29787576

Bioinformatic and Comparative Localization of Rab Proteins Reveals Functional Insights into the Uncharacterized GTPases Ypt10p and Ypt11p†

PubMed Central

Buvelot Frei, Stéphanie; Rahl, Peter B.; Nussbaum, Maria; Briggs, Benjamin J.; Calero, Monica; Janeczko, Stephanie; Regan, Andrew D.; Chen, Catherine Z.; Barral, Yves; Whittaker, Gary R.; Collins, Ruth N.

2006-01-01

A striking characteristic of a Rab protein is its steady-state localization to the cytosolic surface of a particular subcellular membrane. In this study, we have undertaken a combined bioinformatic and experimental approach to examine the evolutionary conservation of Rab protein localization. A comprehensive primary sequence classification shows that 10 out of the 11 Rab proteins identified in the yeast (Saccharomyces cerevisiae) genome can be grouped within a major subclass, each comprising multiple Rab orthologs from diverse species. We compared the locations of individual yeast Rab proteins with their localizations following ectopic expression in mammalian cells. Our results suggest that green fluorescent protein-tagged Rab proteins maintain localizations across large evolutionary distances and that the major known player in the Rab localization pathway, mammalian Rab-GDI, is able to function in yeast. These findings enable us to provide insight into novel gene functions and classify the uncharacterized Rab proteins Ypt10p (YBR264C) as being involved in endocytic function and Ypt11p (YNL304W) as being localized to the endoplasmic reticulum, where we demonstrate it is required for organelle inheritance. PMID:16980630

BAG3 Expression in Glioblastoma Cells Promotes Accumulation of Ubiquitinated Clients in an Hsp70-dependent Manner*

PubMed Central

Gentilella, Antonio; Khalili, Kamel

2011-01-01

Disposal of damaged proteins and protein aggregates is a prerequisite for the maintenance of cellular homeostasis and impairment of this disposal can lead to a broad range of pathological conditions, most notably in brain-associated disorders including Parkinson and Alzheimer diseases, and cancer. In this respect, the Protein Quality Control (PQC) pathway plays a central role in the clearance of damaged proteins. The Hsc/Hsp70-co-chaperone BAG3 has been described as a new and critical component of the PQC in several cellular contexts. For example, the expression of BAG3 in the rodent brain correlates with the engagement of protein degradation machineries in response to proteotoxic stress. Nevertheless, little is known about the molecular events assisted by BAG3. Here we show that ectopic expression of BAG3 in glioblastoma cells leads to the activation of an HSF1-driven stress response, as attested by transcriptional activation of BAG3 and Hsp70. BAG3 overexpression determines an accumulation of ubiquitinated proteins and this event requires the N-terminal region, WW domain of BAG3 and the association of BAG3 with Hsp70. The ubiquitination mainly occurs on BAG3-client proteins and the inhibition of proteasomal activity results in a further accumulation of ubiquitinated clients. At the cellular level, overexpression of BAG3 in glioblastoma cell lines, but not in non-glial cells, results in a remarkable decrease in colony formation capacity and this effect is reverted when the binding of BAG3 to Hsp70 is impaired. These observations provide the first evidence for an involvement of BAG3 in the ubiquitination and turnover of its partners. PMID:21233200

Myxoma Virus Expressing Interleukin-15 Fails To Cause Lethal Myxomatosis in European Rabbits▿

PubMed Central

Liu, Jia; Wennier, Sonia; Reinhard, Mary; Roy, Edward; MacNeill, Amy; McFadden, Grant

2009-01-01

Myxoma virus (MYXV) is a poxvirus pathogenic only for European rabbits, but its permissiveness in human cancer cells gives it potential as an oncolytic virus. A recombinant MYXV expressing both the tdTomato red fluorescent protein and interleukin-15 (IL-15) (vMyx-IL-15-tdTr) was constructed. Cells infected with vMyx-IL-15-tdTr secreted bioactive IL-15 and had in vitro replication kinetics similar to that of wild-type MYXV. To determine the safety of this virus for future oncolytic studies, we tested its pathogenesis in European rabbits. In vivo, vMyx-IL-15-tdTr no longer causes lethal myxomatosis. Thus, ectopic IL-15 functions as an antiviral cytokine in vivo, and vMyx-IL-15-tdTr is a safe candidate for animal studies of oncolytic virotherapy. PMID:19279088

Myxoma virus expressing interleukin-15 fails to cause lethal myxomatosis in European rabbits.

PubMed

Liu, Jia; Wennier, Sonia; Reinhard, Mary; Roy, Edward; MacNeill, Amy; McFadden, Grant

2009-06-01

Myxoma virus (MYXV) is a poxvirus pathogenic only for European rabbits, but its permissiveness in human cancer cells gives it potential as an oncolytic virus. A recombinant MYXV expressing both the tdTomato red fluorescent protein and interleukin-15 (IL-15) (vMyx-IL-15-tdTr) was constructed. Cells infected with vMyx-IL-15-tdTr secreted bioactive IL-15 and had in vitro replication kinetics similar to that of wild-type MYXV. To determine the safety of this virus for future oncolytic studies, we tested its pathogenesis in European rabbits. In vivo, vMyx-IL-15-tdTr no longer causes lethal myxomatosis. Thus, ectopic IL-15 functions as an antiviral cytokine in vivo, and vMyx-IL-15-tdTr is a safe candidate for animal studies of oncolytic virotherapy.

Prohibitin (PHB) inhibits apoptosis in rat granulosa cells (GCs) through the extracellular signal-regulated kinase 1/2 (ERK1/2) and the Bcl family of proteins.

PubMed

Chowdhury, Indrajit; Thompson, Winston E; Welch, Crystal; Thomas, Kelwyn; Matthews, Roland

2013-12-01

Mammalian ovarian follicular development is tightly regulated by crosstalk between cell death and survival signals, which include both endocrine and intra-ovarian regulators. Whether the follicle ultimately ovulates or undergoes atresia is dependent on the expression and actions of factors promoting follicular cell proliferation, differentiation or apoptosis. Prohibitin (PHB) is a highly conserved, ubiquitous protein that is abundantly expressed in granulosa cells (GCs) and associated with GC differentiation and apoptosis. The current study was designed to characterize the regulation of anti-apoptotic and pro-apoptotic factors in undifferentiated rat GCs (gonadotropin independent phase) governed by PHB. Microarray technology was initially employed to identify potential apoptosis-related genes, whose expression levels within GCs were altered by either staurosporine (STS) alone or STS in presence of ectopically over-expressed PHB. Next, immunoblot studies were performed to examine the expression patterns of selective Bcl-2 family members identified by the microarray analysis, which are commonly regulated in the intrinsic-apoptotic pathway. These studies were designed to measure protein levels of Bcl2 family in relation to expression of the acidic isoform (phosphorylated) PHB and the components of MEK-Erk1/2 pathway. These studies indicated that over-expression of PHB in undifferentiated GCs inhibit apoptosis which concomitantly results in an increased level of the anti-apoptotic proteins Bcl2 and Bclxl, reduced release of cytochrome c from mitochondria and inhibition of caspase-3 activity. In contrast, silencing of PHB expression resulted in change of mitochondrial morphology from the regular reticular network to a fragmented form, which enhanced sensitization of these GCs to the induction of apoptosis. Collectively, these studies have provided new insights on the PHB-mediated anti-apoptotic mechanism, which occurs in undifferentiated GCs through a PHB → Mek-Erk1/2 → Bcl/Bcl-xL pathway and may have important clinical implications.

Ebola virus proteins NP, VP35, and VP24 are essential and sufficient to mediate nucleocapsid transport.

PubMed

Takamatsu, Yuki; Kolesnikova, Larissa; Becker, Stephan

2018-01-30

The intracytoplasmic movement of nucleocapsids is a crucial step in the life cycle of enveloped viruses. Determination of the viral components necessary for viral nucleocapsid transport competency is complicated by the dynamic and complex nature of nucleocapsid assembly and the lack of appropriate model systems. Here, we established a live-cell imaging system based on the ectopic expression of fluorescent Ebola virus (EBOV) fusion proteins, allowing the visualization and analysis of the movement of EBOV nucleocapsid-like structures with different protein compositions. Only three of the five EBOV nucleocapsid proteins-nucleoprotein, VP35, and VP24-were necessary and sufficient to form transport-competent nucleocapsid-like structures. The transport of these structures was found to be dependent on actin polymerization and to have dynamics that were undistinguishable from those of nucleocapsids in EBOV-infected cells. The intracytoplasmic movement of nucleocapsid-like structures was completely independent of the viral matrix protein VP40 and the viral surface glycoprotein GP. However, VP40 greatly enhanced the efficiency of nucleocapsid recruitment into filopodia, the sites of EBOV budding.

The homeobox gene mirror links EGF signalling to embryonic dorso-ventral axis formation through notch activation.

PubMed

Jordan, K C; Clegg, N J; Blasi, J A; Morimoto, A M; Sen, J; Stein, D; McNeill, H; Deng, W M; Tworoger, M; Ruohola-Baker, H

2000-04-01

Recent studies in vertebrates and Drosophila melanogaster have revealed that Fringe-mediated activation of the Notch pathway has a role in patterning cell layers during organogenesis. In these processes, a homeobox-containing transcription factor is responsible for spatially regulating fringe (fng) expression and thus directing activation of the Notch pathway along the fng expression border. Here we show that this may be a general mechanism for patterning epithelial cell layers. At three stages in Drosophila oogenesis, mirror (mirr) and fng have complementary expression patterns in the follicle-cell epithelial layer, and at all three stages loss of mirr enlarges, and ectopic expression of mirr restricts, fng expression, with consequences for follicle-cell patterning. These morphological changes are similar to those caused by Notch mutations. Ectopic expression of mirr in the posterior follicle cells induces a stripe of rhomboid (rho) expression and represses pipe (pip), a gene with a role in the establishment of the dorsal-ventral axis, at a distance. Ectopic Notch activation has a similar long-range effect on pip. Our results suggest that Mirror and Notch induce secretion of diffusible morphogens and we have identified TGF-beta (encoded by dpp) as such a molecule in germarium. We also found that mirr expression in dorsal follicle cells is induced by the EGF-receptor (EGFR) pathway and that mirr then represses pip expression in all but the ventral follicle cells, connecting EGFR activation in the dorsal follicle cells to repression of pip in the dorsal and lateral follicle cells. Our results suggest that the differentiation of ventral follicle cells is not a direct consequence of germline signalling, but depends on long-range signals from dorsal follicle cells, and provide a link between early and late events in Drosophila embryonic dorsal-ventral axis formation.

Induction of miR-137 by Isorhapontigenin (ISO) Directly Targets Sp1 Protein Translation and Mediates Its Anticancer Activity Both In Vitro and In Vivo.

PubMed

Zeng, Xingruo; Xu, Zhou; Gu, Jiayan; Huang, Haishan; Gao, Guangxun; Zhang, Xiaoru; Li, Jingxia; Jin, Honglei; Jiang, Guosong; Sun, Hong; Huang, Chuanshu

2016-03-01

Our recent studies found that isorhapontigenin (ISO) showed a significant inhibitory effect on human bladder cancer cell growth, accompanied with cell-cycle G0-G1 arrest as well as downregulation of Cyclin D1 expression at transcriptional level via inhibition of Sp1 transactivation in bladder cancer cells. In the current study, the potential ISO inhibition of bladder tumor formation has been explored in a xenograft nude mouse model, and the molecular mechanisms underlying ISO inhibition of Sp1 expression and anticancer activities have been elucidated both in vitro and in vivo. Moreover, the studies demonstrated that ISO treatment induced the expression of miR-137, which in turn suppressed Sp1 protein translation by directly targeting Sp1 mRNA 3'-untranslated region (UTR). Similar to ISO treatment, ectopic expression of miR-137 alone led to G0-G1 cell growth arrest and inhibition of anchorage-independent growth in human bladder cancer cells, which could be completely reversed by overexpression of GFP-Sp1. The inhibition of miR-137 expression attenuated ISO-induced inhibition of Sp1/Cyclin D1 expression, induction of G0-G1 cell growth arrest, and suppression of cell anchorage-independent growth. Taken together, our studies have demonstrated that miR-137 induction by ISO targets Sp1 mRNA 3'-UTR and inhibits Sp1 protein translation, which consequently results in reduction of Cyclin D1 expression, induction of G0-G1 growth arrest, and inhibition of anchorage-independent growth in vitro and in vivo. Our results have provided novel insights into understanding the anticancer activity of ISO in the therapy of human bladder cancer. ©2016 American Association for Cancer Research.

MSH1 Is a Plant Organellar DNA Binding and Thylakoid Protein under Precise Spatial Regulation to Alter Development

DOE PAGES

Virdi, Kamaldeep S.; Wamboldt, Yashitola; Kundariya, Hardik; ...

2015-11-14

As metabolic centers, plant organelles participate in maintenance, defense, and signaling. MSH1 is a plant-specific protein involved in organellar genome stability in mitochondria and plastids. Plastid depletion of MSH1 causes heritable, non-genetic changes in development and DNA methylation. We investigated the msh1 phenotype using hemi-complementation mutants and transgene-null segregants from RNAi suppression lines to sub-compartmentalize MSH1 effects. We show that MSH1 expression is spatially regulated, specifically localizing to plastids within the epidermis and vascular parenchyma. The protein binds DNA and localizes to plastid and mitochondrial nucleoids, but fractionation and protein–protein interactions data indicate that MSH1 also associates with the thylakoidmore » membrane. Plastid MSH1 depletion results in variegation, abiotic stress tolerance, variable growth rate, and delayed maturity. Depletion from mitochondria results in 7%–10% of plants altered in leaf morphology, heat tolerance, and mitochondrial genome stability. MSH1 does not localize within the nucleus directly, but plastid depletion produces non-genetic changes in flowering time, maturation, and growth rate that are heritable independent of MSH1. MSH1 depletion alters non-photoactive redox behavior in plastids and a sub-set of mitochondrially altered lines. Ectopic expression produces deleterious effects, underlining its strict expression control. Unraveling the complexity of the MSH1 effect offers insight into triggers of plant-specific, transgenerational adaptation behaviors.« less

MazEF toxin-antitoxin proteins alter Escherichia coli cell morphology and infrastructure during persister formation and regrowth.

PubMed

Cho, Junho; Carr, Anita Nicole; Whitworth, Lisa; Johnson, Brent; Wilson, Kevin Scott

2017-03-01

When exposed to antibiotics, many bacteria respond by activating intracellular 'toxin' proteins, which arrest cell growth and induce formation of persister cells that survive antibiotics. After antibiotics are removed, persisters can regrow by synthesizing 'antitoxin' proteins that sequester toxin proteins. In Escherichia coli, MazE antitoxin sequesters the activity of MazF toxin, which extensively cleaves cellular RNAs. Although the functions of MazEF proteins are well characterized, there is surprisingly little known about their effects on cell structure. Here, using a combination of microscopy techniques, we visualized the effects of MazEF and three bactericidal antibiotics on E. coli cell morphology and infrastructure. When ectopically expressed in E. coli, MazF temporarily stalled cell growth and induced persister formation, but only mildly elevated DNA mutagenesis. Viewed by electron microscopy, MazF-expressing persister cells were arrested in cell growth and division. Their chromosomal DNAs were compacted into thread-like structures. Their ribosomes were excluded from their nucleoids. After exposure to ciprofloxacin, persister regrowth was activated by MazE. Cell division remained inhibited while cells became extraordinarily elongated, then divided multiple times during stationary growth phase. This extreme filamentation during persister regrowth was unique to ciprofloxacin-treated persisters, likely caused by inhibition of cell division during regrowth, and was not observed with kanamycin-treated persisters.

CIKS, a connection to IκB kinase and stress-activated protein kinase

PubMed Central

Leonardi, Antonio; Chariot, Alain; Claudio, Estefania; Cunningham, Kirk; Siebenlist, Ulrich

2000-01-01

Pathogens, inflammatory signals, and stress cause acute transcriptional responses in cells. The induced expression of genes in response to these signals invariably involves transcription factors of the NF-κB and AP-1/ATF families. Activation of NF-κB factors is thought to be mediated primarily via IκB kinases (IKK), whereas that of AP-1/ATF can be mediated by stress-activated protein kinases (SAPKs; also named Jun kinases or JNKs). IKKα and IKKβ are two catalytic subunits of a core IKK complex that also contains the regulatory subunit NEMO (NF-κB essential modulator)/IKKγ. The latter protein is essential for activation of the IKKs, but its mechanism of action is not known. Here we describe the molecular cloning of CIKS (connection to IKK and SAPK/JNK), a previously unknown protein that directly interacts with NEMO/IKKγ in cells. When ectopically expressed, CIKS stimulates IKK and SAPK/JNK kinases and it transactivates an NF-κB-dependent reporter. Activation of NF-κB is prevented in the presence of kinase-deficient, interfering mutants of the IKKs. CIKS may help to connect upstream signaling events to IKK and SAPK/JNK modules. CIKS could coordinate the activation of two stress-induced signaling pathways, functions reminiscent of those noted for tumor necrosis factor receptor-associated factor adaptor proteins. PMID:10962033

Specification of spatial identities of cerebellar neuron progenitors by ptf1a and atoh1 for proper production of GABAergic and glutamatergic neurons.

PubMed

Yamada, Mayumi; Seto, Yusuke; Taya, Shinichiro; Owa, Tomoo; Inoue, Yukiko U; Inoue, Takayoshi; Kawaguchi, Yoshiya; Nabeshima, Yo-Ichi; Hoshino, Mikio

2014-04-02

In the cerebellum, the bHLH transcription factors Ptf1a and Atoh1 are expressed in distinct neuroepithelial regions, the ventricular zone (VZ) and the rhombic lip (RL), and are required for producing GABAergic and glutamatergic neurons, respectively. However, it is unclear whether Ptf1a or Atoh1 is sufficient for specifying GABAergic or glutamatergic neuronal fates. To test this, we generated two novel knock-in mouse lines, Ptf1a(Atoh1) and Atoh1(Ptf1a), that are designed to express Atoh1 and Ptf1a ectopically in the VZ and RL, respectively. In Ptf1a(Atoh1) embryos, ectopically Atoh1-expressing VZ cells produced glutamatergic neurons, including granule cells and deep cerebellar nuclei neurons. Correspondingly, in Atoh1(Ptf1a) animals, ectopically Ptf1a-expressing RL cells produced GABAergic populations, such as Purkinje cells and GABAergic interneurons. Consistent results were also obtained from in utero electroporation of Ptf1a or Atoh1 into embryonic cerebella, suggesting that Ptf1a and Atoh1 are essential and sufficient for GABAergic versus glutamatergic specification in the neuroepithelium. Furthermore, birthdating analyses with BrdU in the knock-in mice or with electroporation studies showed that ectopically produced fate-changed neuronal types were generated at temporal schedules closely simulating those of the wild-type RL and VZ, suggesting that the VZ and RL share common temporal information. Observations of knock-in brains as well as electroporated brains revealed that Ptf1a and Atoh1 mutually negatively regulate their expression, probably contributing to formation of non-overlapping neuroepithelial domains. These findings suggest that Ptf1a and Atoh1 specify spatial identities of cerebellar neuron progenitors in the neuroepithelium, leading to appropriate production of GABAergic and glutamatergic neurons, respectively.

Repression of c-Kit by p53 is mediated by miR-34 and is associated with reduced chemoresistance, migration and stemness

PubMed Central

Siemens, Helge; Jackstadt, Rene; Kaller, Markus; Hermeking, Heiko

2013-01-01

The c-Kit receptor tyrosine kinase is commonly over-expressed in different types of cancer. p53 activation is known to result in the down-regulation of c-Kit. However, the underlying mechanism has remained unknown. Here, we show that the p53-induced miR-34 microRNA family mediates repression of c-Kit by p53 via a conserved seed-matching sequence in the c-Kit 3'-UTR. Ectopic miR-34a resulted in a decrease in Erk signaling and transformation, which was dependent on the down-regulation of c-Kit expression. Furthermore, ectopic expression of c-Kit conferred resistance of colorectal cancer (CRC) cells to treatment with 5-fluorouracil (5-FU), whereas ectopic miR-34a sensitized the cells to 5-FU. After stimulation with c-Kit ligand/stem cell factor (SCF) Colo320 CRC cells displayed increased migration/invasion, whereas ectopic miR-34a inhibited SCF-induced migration/invasion. Activation of a conditional c-Kit allele induced several stemness markers in DLD-1 CRC cells. In primary CRC samples elevated c-Kit expression also showed a positive correlation with markers of stemness, such as Lgr5, CD44, OLFM4, BMI-1 and β-catenin. On the contrary, activation of a conditional miR-34a allele in DLD-1 cells diminished the expression of c-Kit and several stemness markers (CD44, Lgr5 and BMI-1) and suppressed sphere formation. MiR-34a also suppressed enhanced sphere-formation after exposure to SCF. Taken together, our data establish c-Kit as a new direct target of miR-34 and demonstrate that this regulation interferes with several c-Kit-mediated effects on cancer cells. Therefore, this regulation may be potentially relevant for future diagnostic and therapeutic approaches. PMID:24009080

Tissue-Specific Gain of RTK Signalling Uncovers Selective Cell Vulnerability during Embryogenesis

PubMed Central

Audebert, Stéphane; Helmbacher, Françoise; Dono, Rosanna; Maina, Flavio

2015-01-01

The successive events that cells experience throughout development shape their intrinsic capacity to respond and integrate RTK inputs. Cellular responses to RTKs rely on different mechanisms of regulation that establish proper levels of RTK activation, define duration of RTK action, and exert quantitative/qualitative signalling outcomes. The extent to which cells are competent to deal with fluctuations in RTK signalling is incompletely understood. Here, we employ a genetic system to enhance RTK signalling in a tissue-specific manner. The chosen RTK is the hepatocyte growth factor (HGF) receptor Met, an appropriate model due to its pleiotropic requirement in distinct developmental events. Ubiquitously enhanced Met in Cre/loxP-based Rosa26 stopMet knock-in context (Del-R26 Met) reveals that most tissues are capable of buffering enhanced Met-RTK signalling thus avoiding perturbation of developmental programs. Nevertheless, this ubiquitous increase of Met does compromise selected programs such as myoblast migration. Using cell-type specific Cre drivers, we genetically showed that altered myoblast migration results from ectopic Met expression in limb mesenchyme rather than in migrating myoblasts themselves. qRT-PCR analyses show that ectopic Met in limbs causes molecular changes such as downregulation in the expression levels of Notum and Syndecan4, two known regulators of morphogen gradients. Molecular and functional studies revealed that ectopic Met expression in limb mesenchyme does not alter HGF expression patterns and levels, but impairs HGF bioavailability. Together, our findings show that myoblasts, in which Met is endogenously expressed, are capable of buffering increased RTK levels, and identify mesenchymal cells as a cell type vulnerable to ectopic Met-RTK signalling. These results illustrate that embryonic cells are sensitive to alterations in the spatial distribution of RTK action, yet resilient to fluctuations in signalling levels of an RTK when occurring in its endogenous domain of activity. PMID:26393505

MiR-135 post-transcriptionally regulates FOXO1 expression and promotes cell proliferation in human malignant melanoma cells.

PubMed

Ren, Jian-Wen; Li, Zhang-Jun; Tu, Chen

2015-01-01

Malignant melanoma is the deadliest form of all skin cancers. Recently, microRNAs (miRNAs) are small, non-coding RNAs that regulate gene expression by targeted repression of transcription and translation and play essential roles during cancer development. Our study showed that miR-135a is upregulated in malignant melanoma tissues and cell lines by using Real-time PCR assay. Enforced expression of miR-135a in malignant melanoma cells promotes cell proliferation, tumorigenicity, and cell cycle progression, whereas inhibition of miR-135a reverses the function. Additionally, we demonstrated FOXO1 is a direct target of miR-135a and transcriptionally down-regulated by miR-135a. Ectopic expression of miR-135a led to downregulation of the FOXO1 protein, resulting in upregulation of Cyclin D1, and downregulation of P21(Cip1) and P27(Kip1) through AKT pathway. Our findings suggested that miR-135a represents a potential onco-miRNA and plays an important role in malignant melanoma progression by suppressing FOXO1 expression.

Ectopic expression of AtPAD4 broadens resistance of soybean to soybean cyst and root-knot nematodes.

PubMed

Youssef, Reham M; MacDonald, Margaret H; Brewer, Eric P; Bauchan, Gary R; Kim, Kyung-Hwan; Matthews, Benjamin F

2013-04-25

The gene encoding PAD4 (PHYTOALEXIN-DEFICIENT4) is required in Arabidopsis for expression of several genes involved in the defense response to Pseudomonas syringae pv. maculicola. AtPAD4 (Arabidopsis thaliana PAD4) encodes a lipase-like protein that plays a regulatory role mediating salicylic acid signaling. We expressed the gene encoding AtPAD4 in soybean roots of composite plants to test the ability of AtPAD4 to deter plant parasitic nematode development. The transformed roots were challenged with two different plant parasitic nematode genera represented by soybean cyst nematode (SCN; Heterodera glycines) and root-knot nematode (RKN; Meloidogyne incognita). Expression of AtPAD4 in soybean roots decreased the number of mature SCN females 35 days after inoculation by 68 percent. Similarly, soybean roots expressing AtPAD4 exhibited 77 percent fewer galls when challenged with RKN. Our experiments show that AtPAD4 can be used in an economically important crop, soybean, to provide a measure of resistance to two different genera of nematodes.

The Arabidopsis BRAHMA Chromatin-Remodeling ATPase Is Involved in Repression of Seed Maturation Genes in Leaves1[W][OA

PubMed Central

Tang, Xurong; Hou, Anfu; Babu, Mohan; Nguyen, Vi; Hurtado, Lidia; Lu, Qing; Reyes, Jose C.; Wang, Aiming; Keller, Wilfred A.; Harada, John J.; Tsang, Edward W.T.; Cui, Yuhai

2008-01-01

Synthesis and accumulation of seed storage proteins (SSPs) is an important aspect of the seed maturation program. Genes encoding SSPs are specifically and highly expressed in the seed during maturation. However, the mechanisms that repress the expression of these genes in leaf tissue are not well understood. To gain insight into the repression mechanisms, we performed a genetic screen for mutants that express SSPs in leaves. Here, we show that mutations affecting BRAHMA (BRM), a SNF2 chromatin-remodeling ATPase, cause ectopic expression of a subset of SSPs and other embryogenesis-related genes in leaf tissue. Consistent with the notion that such SNF2-like ATPases form protein complexes in vivo, we observed similar phenotypes for mutations of AtSWI3C, a BRM-interacting partner, and BSH, a SNF5 homolog and essential SWI/SNF subunit. Chromatin immunoprecipitation experiments show that BRM is recruited to the promoters of a number of embryogenesis genes in wild-type leaves, including the 2S genes, expressed in brm leaves. Consistent with its role in nucleosome remodeling, BRM appears to affect the chromatin structure of the At2S2 promoter. Thus, the BRM-containing chromatin-remodeling ATPase complex involved in many aspects of plant development mediates the repression of SSPs in leaf tissue. PMID:18508955

The cold-induced basic helix-loop-helix transcription factor gene MdCIbHLH1 encodes an ICE-like protein in apple

PubMed Central

2012-01-01

Background Plant growth is greatly affected by low temperatures, and the expression of a number of genes is induced by cold stress. Although many genes in the cold signaling pathway have been identified in Arabidopsis, little is known about the transcription factors involved in the cold stress response in apple. Results Here, we show that the apple bHLH (basic helix-loop-helix) gene MdCIbHLH1 (Cold-Induced bHLH1), which encodes an ICE-like protein, was noticeably induced in response to cold stress. The MdCIbHLH1 protein specifically bound to the MYC recognition sequences in the AtCBF3 promoter, and MdCIbHLH1 overexpression enhanced cold tolerance in transgenic Arabidopsis. In addition, the MdCIbHLH1 protein bound to the promoters of MdCBF2 and favorably contributed to cold tolerance in transgenic apple plants by upregulating the expression of MdCBF2 through the CBF (C-repeat-binding factor) pathway. Our findings indicate that MdCIbHLH1 functions in stress tolerance in different species. For example, ectopic MdCIbHLH1 expression conferred enhanced chilling tolerance in transgenic tobacco. Finally, we observed that cold induces the degradation of the MdCIbHLH1 protein in apple and that this degradation was potentially mediated by ubiquitination and sumoylation. Conclusions Based on these findings, MdCIbHLH1 encodes a transcription factor that is important for the cold tolerance response in apple. PMID:22336381

The Zinc-finger protein ASCIZ regulates B cell development via DYNLL1 and Bim

PubMed Central

Jurado, Sabine; Gleeson, Kimberly; O’Donnell, Kristy; Izon, David J.; Walkley, Carl R.; Strasser, Andreas; Tarlinton, David M.

2012-01-01

Developing B lymphocytes expressing defective or autoreactive pre-B or B cell receptors (BCRs) are eliminated by programmed cell death, but how the balance between death and survival signals is regulated to prevent immunodeficiency and autoimmunity remains incompletely understood. In this study, we show that absence of the essential ATM (ataxia telangiectasia mutated) substrate Chk2-interacting Zn2+-finger protein (ASCIZ; also known as ATMIN/ZNF822), a protein with dual functions in the DNA damage response and as a transcription factor, leads to progressive cell loss from the pre-B stage onwards and severely diminished splenic B cell numbers in mice. This lymphopenia cannot be suppressed by deletion of p53 or complementation with a prearranged BCR, indicating that it is not caused by impaired DNA damage responses or defective V(D)J recombination. Instead, ASCIZ-deficient B cell precursors contain highly reduced levels of DYNLL1 (dynein light chain 1; LC8), a recently identified transcriptional target of ASCIZ, and normal B cell development can be restored by ectopic Dynll1 expression. Remarkably, the B cell lymphopenia in the absence of ASCIZ can also be fully suppressed by deletion of the proapoptotic DYNLL1 target Bim. Our findings demonstrate a key role for ASCIZ in regulating the survival of developing B cells by activating DYNLL1 expression, which may then modulate Bim-dependent apoptosis. PMID:22891272

Effect of HIV-1 Tat on the formation of the mitotic spindle by interaction with ribosomal protein S3.

PubMed

Kim, Jiyoung; Kim, Yeon-Soo

2018-06-06

Human immunodeficiency virus type 1 (HIV-1) Tat, an important regulator of viral transcription, interacts with diverse cellular proteins and promotes or inhibits cell proliferation. Here, we show that ribosomal protein S3 (RPS3) plays an important role in mitosis through an interaction with α-tubulin and that Tat binds to and inhibits the localization of RPS3 in the mitotic spindle during mitosis. RPS3 colocalized with α-tubulin around chromosomes in the mitotic spindle. Depletion of RPS3 promoted α-tubulin assembly, while overexpression of RPS3 impaired α-tubulin assembly. Depletion of RPS3 resulted in aberrant mitotic spindle formation, segregation failure, and defective abscission. Moreover, ectopic expression of RPS3 rescued the cell proliferation defect in RPS3-knockdown cells. HIV-1 Tat interacted with RPS3 through its basic domain and increased the level of RPS3 in the nucleus. Expression of Tat caused defects in mitotic spindle formation and chromosome assembly in mitosis. Moreover, the localization of RPS3 in the mitotic spindle was disrupted when HIV-1 Tat was expressed in HeLa and Jurkat cells. These results suggest that Tat inhibits cell proliferation via an interaction with RPS3 and thereby disrupts mitotic spindle formation during HIV-1 infection. These results might provide insight into the mechanism underlying lymphocyte pathogenesis during HIV-1 infection.

Role of zinc-finger anti-viral protein in host defense against Sindbis virus

PubMed Central

Kozaki, Tatsuya; Takahama, Michihiro; Misawa, Takuma; Matsuura, Yoshiharu; Saitoh, Tatsuya

2015-01-01

Accumulating evidence indicates that type I interferon (IFN) mediates the host protective response to RNA viruses. However, the anti-viral effector molecules involved in this response have not been fully identified. Here, we show that zinc-finger anti-viral protein (ZAP), an IFN-inducible gene, plays a critical role in the elimination of Sindbis virus (SINV) in vitro and in vivo. The loss of ZAP greatly enhances the replication of SINV but does not inhibit type I IFN production in primary mouse embryonic fibroblasts (MEFs). ZAP binds and destabilizes SINV RNA, thereby suppressing the replication of SINV. Type I IFN fails to suppress SINV replication in ZAP-deficient MEFs, whereas the ectopic expression of ZAP is sufficient to suppress the replication of SINV in MEFs lacking the expression of type I IFN and the IFN-inducible genes. ZAP-deficient mice are highly susceptible to SINV infection, although they produce sufficient amounts of type I IFN. Therefore, ZAP is an RNA-sensing anti-viral effector molecule that mediates the type-I-IFN-dependent host defense against SINV. PMID:25758257

Genotoxic stress-induced activation of Plk3 is partly mediated by Chk2.

PubMed

Xie, Suqing; Wu, Huiyun; Wang, Qi; Kunicki, Jan; Thomas, Raymond O; Hollingsworth, Robert E; Cogswell, John; Dai, Wei

2002-01-01

Polo-like kinase 3 (Plk3, alternatively termed Prk) is involved in the regulation of DNA damage checkpoint as well as in M-phase function. Plk3 physically interacts with p53 and phosphorylates this tumor suppressor protein on serine-20, suggesting that the role of Plk3 in cell cycle progression is mediated, at least in part, through direct regulation of p53. Here we show that Plk3 is rapidly activated by reactive oxygen species in normal diploid fibroblast cells (WI-38), correlating with a subsequent increase in p53 protein level. Plk3 physically interacts with Chk2 and the interaction is enhanced upon DNA damage. In addition, Chk2 immunoprecipitated from cell lysates of Daudi (which expressed little Plk3) is capable of stimulating the kinase activity of purified recombinant Plk3 in vitro, and this stimulation is more pronounced when Plk3 is supplemented with Chk2 immunoprecipitated from Daudi after DNA damage. Furthermore, ectopic expression Chk2 activates cellular Plk3. Together, our studies suggest Chk2 may mediate direct activation of Plk3 in response to genotoxic stresses.

Spi-C has opposing effects to PU.1 on gene expression in progenitor B cells.

PubMed

Schweitzer, Brock L; Huang, Kelly J; Kamath, Meghana B; Emelyanov, Alexander V; Birshtein, Barbara K; DeKoter, Rodney P

2006-08-15

The Ets transcription factor Spi-C, expressed in B cells and macrophages, is closely related to PU.1 and has the ability to recognize the same DNA consensus sequence. However, the function of Spi-C has yet to be determined. The purpose of this study is to further examine Spi-C activity in B cell development. First, using retroviral vectors to infect PU.1(-/-) fetal liver progenitors, Spi-C was found to be inefficient at inducing cytokine-dependent proliferation and differentiation of progenitor B (pro-B) cells or macrophages relative to PU.1 or Spi-B. Next, Spi-C was ectopically expressed in fetal liver-derived, IL-7-dependent pro-B cell lines. Wild-type (WT) pro-B cells ectopically expressing Spi-C (WT-Spi-C) have several phenotypic characteristics of pre-B cells such as increased CD25 and decreased c-Kit surface expression. In addition, WT-Spi-C pro-B cells express increased levels of IgH sterile transcripts and reduced levels of expression and transcription of the FcgammaRIIb gene. Gel-shift analysis suggests that Spi-C, ectopically expressed in pro-B cells, can bind PU.1 consensus sites in the IgH intronic enhancer and FcgammaRIIb promoter. Transient transfection analysis demonstrated that PU.1 functions to repress the IgH intronic enhancer and activate the FcgammaRIIb promoter, while Spi-C opposes these activities. WT-Spi-C pro-B cells have reduced levels of dimethylation on lysine 9 of histone H3 within the IgH 3' regulatory region, indicating that Spi-C can contribute to removal of repressive features in the IgH locus. Overall, these studies suggest that Spi-C may promote B cell differentiation by modulating the activity of PU.1-dependent genes.

Treatment of collagenase-induced osteoarthritis with a viral vector encoding TSG-6 results in ectopic bone formation.

PubMed

Broeren, Mathijs G A; Di Ceglie, Irene; Bennink, Miranda B; van Lent, Peter L E M; van den Berg, Wim B; Koenders, Marije I; Blaney Davidson, Esmeralda N; van der Kraan, Peter M; van de Loo, Fons A J

2018-01-01

Tumor necrosis factor-inducible gene 6 (TSG-6) has anti-inflammatory and chondroprotective effects in mouse models of inflammatory arthritis. Because cartilage damage and inflammation are also observed in osteoarthritis (OA), we determined the effect of viral overexpression of TSG-6 in experimental osteoarthritis. Bone marrow-derived cells were differentiated to multinucleated osteoclasts in the presence of recombinant TSG-6 or after transduction with a lentiviral TSG-6 expression vector. Multi-nucleated osteoclasts were analyzed after tartrate resistant acid phosphatase staining and resorption activity was determined on dentin slices. Collagenase-induced osteoarthritis (CIOA) was induced in C57BL/6 mice after intra-articular injection of an adenoviral TSG-6 or control luciferase expression vector. Inflammation-related protease activity was measured using bioluminescent Prosense probes. After a second adenovirus injection, cartilage damage was assessed in histological sections stained with Safranin-O. Ectopic bone formation was scored in X-ray images of the affected knees. TSG-6 did not inhibit the formation of multi-nucleated osteoclasts, but caused a significant reduction in the resorption activity on dentin slices. Adenoviral TSG-6 gene therapy in CIOA could not reduce the cartilage damage compared to the luciferase control virus and no significant difference in inflammation-related protease activity was noted between the TSG-6 and control treated group. Instead, X-ray analysis and histological analysis revealed the presence of ectopic bone formation in the TSG-6 treated group. Gene therapy based on the expression of TSG-6 could not provide cartilage protection in experimental osteoarthritis, but instead resulted in increased ectopic bone formation.

Analysis of Expression Pattern and Genetic Deletion of Netrin5 in the Developing Mouse

PubMed Central

Garrett, Andrew M.; Jucius, Thomas J.; Sigaud, Liam P. R.; Tang, Fu-Lei; Xiong, Wen-Cheng; Ackerman, Susan L.; Burgess, Robert W.

2016-01-01

Boundary cap cells (BCC) are a transient, neural-crest-derived population found at the motor exit point (MEP) and dorsal root entry zone (DREZ) of the embryonic spinal cord. These cells contribute to the central/peripheral nervous system (CNS/PNS) boundary, and in their absence neurons and glia from the CNS migrate into the PNS. We found Netrin5 (Ntn5), a previously unstudied member of the netrin gene family, to be robustly expressed in BCC. We generated Ntn5 knockout mice and examined neurodevelopmental and BCC-related phenotypes. No abnormalities in cranial nerve guidance, dorsal root organization, or sensory projections were found. However, Ntn5 mutant embryos did have ectopic motor neurons (MNs) that migrated out of the ventral horn and into the motor roots. Previous studies have implicated semaphorin6A (Sema6A) in BCC signaling to plexinA2 (PlxnA2)/neuropilin2 (Nrp2) in MNs in restricting MN cell bodies to the ventral horn, particularly in the caudal spinal cord. In Ntn5 mutants, ectopic MNs are likely to be a different population, as more ectopias were found rostrally. Furthermore, ectopic MNs in Ntn5 mutants were not immunoreactive for NRP2. The netrin receptor deleted in colorectal cancer (DCC) is a potential receptor for NTN5 in MNs, as similar ectopic neurons were found in Dcc mutant mice, but not in mice deficient for other netrin receptors. Thus, Ntn5 is a novel netrin family member that is expressed in BCC, functioning to prevent MN migration out of the CNS. PMID:26858598

LRIG1, a 3p tumor suppressor, represses EGFR signaling and is a novel epigenetic silenced gene in colorectal cancer

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kou, Changhua, E-mail: chkoukou@hotmail.com; Zhou, Tian; Han, Xilin

2015-08-21

Downregulation of LRIG1 was found in many types of cancer. However, data concerning the possible mechanism of LRIG1 reduction in cancers were not reported yet. To analyze the regulation and function of LRIG1 in colorectal cancer (CRC), 6 cell lines, 46 paired tissues from primary CRC cases were employed in this study. In CRC cell lines, under-expression of LRIG1 was correlated with promoter region hypermethylation, and restoration of LRIG1 was induced by 5-Aza-2'-deoxyazacytidine treatment. Subsequently, we ectopically expressed LRIG1 in LRIG1 low-expressing HCT-116 cells and suppressed LRIG1 in LRIG1 high-expressing LoVo cells. We found that over-expression of LRIG1 inhibits cellmore » proliferation and colony formation and tumor growth, while knockdown of LRIG1 promotes cell proliferation and colony formation. Decreased and increased EGFR/AKT signaling pathway may partially explain the lower and higher rates of proliferation in CRC cells transfected with LRIG1 cDNA or shRNA. In clinical samples, we compared the methylation, mRNA and protein expression of LRIG1 in samples of CRC tissues. A significant increase in LRIG1 methylation was identified in CRC specimens compared to adjacent normal tissues and that it was negatively correlated with its mRNA and protein expression. In conclusion, LRIG1 is frequently methylated in human CRC and consequent mRNA and protein downregulation may contribute to tumor growth by activating EGFR/AKT signaling. - Highlights: • Promoter methylation of LRIG1 occurred in colorectal cancer cells and tumors. • Restoration of LRIG1 inhibits tumor growth in vitro and in vivo. • Overexpression or knockdown of LRIG1 regulates EGFR/AKT and downstream apoptosis. • Methylation of LRIG1 correlates with its mRNA and protein downregulation. • LRIG1 was firstly identified as an epigenetic target in cancer.« less

Homeobox protein TLX3 activates miR-125b expression to promote T-cell acute lymphoblastic leukemia

PubMed Central

Renou, Laurent; Boelle, Pierre-Yves; Deswarte, Caroline; Spicuglia, Salvatore; Benyoucef, Aissa; Calvo, Julien; Uzan, Benjamin; Belhocine, Mohamed; Cieslak, Agata; Landman-Parker, Judith; Baruchel, Andre; Asnafi, Vahid; Pflumio, Françoise; Ballerini, Paola

2017-01-01

The oncogenic mechanisms driven by aberrantly expressed transcription factors in T-cell acute leukemia (T-ALL) are still elusive. MicroRNAs (miRNAs) play an important role in normal development and pathologies. Here, we examined the expression of 738 miRNA species in 41 newly diagnosed pediatric T-ALLs and in human thymus-derived cells. We found that expression of 2 clustered miRNAs, miR-125b/99a, peaks in primitive T cells and is upregulated in the T leukemia homeobox 3 (TLX3)–positive subtype of T-ALL. Using loss- and gain-of-function approaches, we established functional relationships between TLX3 and miR-125b. Both TLX3 and miR-125b support in vitro cell growth and in vivo invasiveness of T-ALL. Besides, ectopic expression of TLX3 or miR-125b in human hematopoietic progenitor cells enhances production of T-cell progenitors and favors their accumulation at immature stages of T-cell development resembling the differentiation arrest observed in TLX3 T-ALL. Ectopic miR-125b also remarkably accelerated leukemia in a xenograft model, suggesting that miR125b is an important mediator of the TLX3-mediated transformation program that takes place in immature T-cell progenitors. Mechanistically, TLX3-mediated activation of miR-125b may impact T-cell differentiation in part via repression of Ets1 and CBFβ genes, 2 regulators of T-lineage. Finally, we established that TLX3 directly regulates miR-125b production through binding and transactivation of LINC00478, a long noncoding RNA gene, which is the host of miR-99a/Let-7c/miR-125b. Altogether, our results reveal an original functional link between TLX3 and oncogenic miR-125b in T-ALL development. PMID:29296717

Myb transcription factors and light regulate sporulation in the oomycete Phytophthora infestans.

PubMed

Xiang, Qijun; Judelson, Howard S

2014-01-01

Life cycle progression in eukaryotic microbes is often influenced by environment. In the oomycete Phytophthora infestans, which causes late blight on potato and tomato, sporangia have been reported to form mostly at night. By growing P. infestans under different light regimes at constant temperature and humidity, we show that light contributes to the natural pattern of sporulation by delaying sporulation until the following dark period. However, illumination does not permanently block sporulation or strongly affect the total number of sporangia that ultimately form. Based on measurements of sporulation-induced genes such as those encoding protein kinase Pks1 and Myb transcription factors Myb2R1 and Myb2R3, it appears that most spore-associated transcripts start to rise four to eight hours before sporangia appear. Their mRNA levels oscillate with the light/dark cycle and increase with the amount of sporangia. An exception to this pattern of expression is Myb2R4, which is induced several hours before the other genes and declines after cultures start to sporulate. Transformants over-expressing Myb2R4 produce twice the number of sporangia and ten-fold higher levels of Myb2R1 mRNA than wild-type, and chromatin immunoprecipitation showed that Myb2R4 binds the Myb2R1 promoter in vivo. Myb2R4 thus appears to be an early regulator of sporulation. We attempted to silence eight Myb genes by DNA-directed RNAi, but succeeded only with Myb2R3, which resulted in suppressed sporulation. Ectopic expression studies of seven Myb genes revealed that over-expression frequently impaired vegetative growth, and in the case of Myb3R6 interfered with sporangia dormancy. We observed that the degree of silencing induced by a hairpin construct was correlated with its copy number, and ectopic expression was often unstable due to epigenetic silencing and transgene excision.

Myb Transcription Factors and Light Regulate Sporulation in the Oomycete Phytophthora infestans

PubMed Central

Xiang, Qijun; Judelson, Howard S.

2014-01-01

Life cycle progression in eukaryotic microbes is often influenced by environment. In the oomycete Phytophthora infestans, which causes late blight on potato and tomato, sporangia have been reported to form mostly at night. By growing P. infestans under different light regimes at constant temperature and humidity, we show that light contributes to the natural pattern of sporulation by delaying sporulation until the following dark period. However, illumination does not permanently block sporulation or strongly affect the total number of sporangia that ultimately form. Based on measurements of sporulation-induced genes such as those encoding protein kinase Pks1 and Myb transcription factors Myb2R1 and Myb2R3, it appears that most spore-associated transcripts start to rise four to eight hours before sporangia appear. Their mRNA levels oscillate with the light/dark cycle and increase with the amount of sporangia. An exception to this pattern of expression is Myb2R4, which is induced several hours before the other genes and declines after cultures start to sporulate. Transformants over-expressing Myb2R4 produce twice the number of sporangia and ten-fold higher levels of Myb2R1 mRNA than wild-type, and chromatin immunoprecipitation showed that Myb2R4 binds the Myb2R1 promoter in vivo. Myb2R4 thus appears to be an early regulator of sporulation. We attempted to silence eight Myb genes by DNA-directed RNAi, but succeeded only with Myb2R3, which resulted in suppressed sporulation. Ectopic expression studies of seven Myb genes revealed that over-expression frequently impaired vegetative growth, and in the case of Myb3R6 interfered with sporangia dormancy. We observed that the degree of silencing induced by a hairpin construct was correlated with its copy number, and ectopic expression was often unstable due to epigenetic silencing and transgene excision. PMID:24704821

Activation of IRE1α-XBP1 pathway induces cell proliferation and invasion in colorectal carcinoma

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jin, Chun; Jin, Zhao; Chen, Nian-zhao

2016-01-29

Cell proliferation and tumor metastasis are considered as the main reasons for death in colorectal carcinoma (CRC). IRE1α-XBP1 pathway is the most conserved UPR pathways, which are activated during ER stress caused by the accumulation of unfolded or misfolded protein in the lumen of ER. Here, we demonstrated the critical role of IRE1α-XBP1 pathway and underlying molecular mechanism in cell proliferation and tumor metastasis in CRC. By the use of tissue microarray analysis of samples from 119 patients with CRC, IRE1α was determined to be an independent predictor of overall survival as higher expression of IRE1α in CRC patients showedmore » lower survival rates (p = 0.0041). RNA interference and ectopic expression of IRE1α were applied to determine the molecular effects of IRE1α in CRC cells. The silencing of IRE1α inhibited the proliferation and blocked the invasion of CRC cells in vitro, while ectopic expression of IRE1α in turn promoted cell proliferation and invasion. IRE1α-XBP1 pathway regulated the mitosis of CRC cells through the directly binding of XBP1s to Cyclin D1 promoter to activate Cyclin D1 expression. Our results reveal that IRE1α-XBP1 pathway plays an important role in tumor progression and epithelial-to-mesenchymal transition (EMT), and IRE1α could be employed as a novel prognostic marker and a promising therapeutic target for CRC. - Highlights: • IRE1 was determined to be an independent predictor of overall survival in CRC patient. • IRE1-XBP1 pathway promoted CRC cell proliferation through regulating Cyclin D1 expression. • IRE1-XBP1 pathway played important role in EMT of CRC cells.« less

ANALYSIS OF TMEFF2 ALLOGRAFTS AND TRANSGENIC MOUSE MODELS REVEALS ROLES IN PROSTATE REGENERATION AND CANCER

PubMed Central

Corbin, JM.; Overcash, RF.; Wren, JD.; Coburn, A.; Tipton, GJ.; Ezzell, JA.; McNaughton, KK.; Fung, KM; Kosanke, SD.; Ruiz-Echevarria, MJ

2015-01-01

BACKGROUND Previous results from our lab indicate a tumor suppressor role for the transmembrane protein with epidermal growth factor and two follistatin motifs 2 (TMEFF2) in prostate cancer (PCa). Here, we further characterize this role and uncover new functions for TMEFF2 in cancer and adult prostate regeneration. METHODS The role of TMEFF2 was examined in PCa cells using Matrigel™ cultures and allograft models of PCa cells. In addition, we developed a transgenic mouse model that expresses TMEFF2 from a prostate specific promoter. Anatomical, histological and metabolic characterizations of the transgenic mouse prostate were conducted. The effect of TMEFF2 in prostate regeneration was studied by analyzing branching morphogenesis in the TMEFF2-expressing mouse lobes and alterations in branching morphogenesis were correlated with the metabolomic profiles of the mouse lobes. The role of TMEFF2 in prostate tumorigenesis in whole animals was investigated by crossing the TMEFF2 transgenic mice with the TRAMP mouse model of PCa and analyzing the histopathological changes in the progeny. RESULTS Ectopic expression of TMEFF2 impairs growth of PCa cells in Matrigel or allograft models. Surprisingly, while TMEFF2 expression in the TRAMP mouse did not have a significant effect on the glandular prostate epithelial lesions, the double TRAMP/TMEFF2 transgenic mice displayed an increased incidence of neuroendocrine type tumors. In addition, TMEFF2 promoted increased branching specifically in the dorsal lobe of the prostate suggesting a potential role in developmental processes. These results correlated with data indicating an alteration in the metabolic profile of the dorsal lobe of the transgenic TMEFF2 mice. CONCLUSIONS Collectively, our results confirm the tumor suppressor role of TMEFF2 and suggest that ectopic expression of TMEFF2 in mouse prostate leads to additional lobe-specific effects in prostate regeneration and tumorigenesis. This points to a complex and multifunctional role for TMEFF2 during PCa progression. PMID:26417683

Analysis of TMEFF2 allografts and transgenic mouse models reveals roles in prostate regeneration and cancer.

PubMed

Corbin, Joshua M; Overcash, Ryan F; Wren, Jonathan D; Coburn, Anita; Tipton, Greg J; Ezzell, Jennifer A; McNaughton, Kirk K; Fung, Kar-Ming; Kosanke, Stanley D; Ruiz-Echevarria, Maria J

2016-01-01

Previous results from our lab indicate a tumor suppressor role for the transmembrane protein with epidermal growth factor and two follistatin motifs 2 (TMEFF2) in prostate cancer (PCa). Here, we further characterize this role and uncover new functions for TMEFF2 in cancer and adult prostate regeneration. The role of TMEFF2 was examined in PCa cells using Matrigel(TM) cultures and allograft models of PCa cells. In addition, we developed a transgenic mouse model that expresses TMEFF2 from a prostate specific promoter. Anatomical, histological, and metabolic characterizations of the transgenic mouse prostate were conducted. The effect of TMEFF2 in prostate regeneration was studied by analyzing branching morphogenesis in the TMEFF2-expressing mouse lobes and alterations in branching morphogenesis were correlated with the metabolomic profiles of the mouse lobes. The role of TMEFF2 in prostate tumorigenesis in whole animals was investigated by crossing the TMEFF2 transgenic mice with the TRAMP mouse model of PCa and analyzing the histopathological changes in the progeny. Ectopic expression of TMEFF2 impairs growth of PCa cells in Matrigel or allograft models. Surprisingly, while TMEFF2 expression in the TRAMP mouse did not have a significant effect on the glandular prostate epithelial lesions, the double TRAMP/TMEFF2 transgenic mice displayed an increased incidence of neuroendocrine type tumors. In addition, TMEFF2 promoted increased branching specifically in the dorsal lobe of the prostate suggesting a potential role in developmental processes. These results correlated with data indicating an alteration in the metabolic profile of the dorsal lobe of the transgenic TMEFF2 mice. Collectively, our results confirm the tumor suppressor role of TMEFF2 and suggest that ectopic expression of TMEFF2 in mouse prostate leads to additional lobe-specific effects in prostate regeneration and tumorigenesis. This points to a complex and multifunctional role for TMEFF2 during PCa progression. © 2015 Wiley Periodicals, Inc.

TRIM25 blockade by RNA interference inhibited migration and invasion of gastric cancer cells through TGF-β signaling.

PubMed

Zhu, Zhenya; Wang, Yong; Zhang, Chunhui; Yu, Shiyong; Zhu, Qi; Hou, Kun; Yan, Bo

2016-01-12

Tripartite Motif Containing 25 (TRIM25), a member of TRIM proteins, has been found abnormally expressed in cancers of female reproductive system. Here, TRIM25 was conspicuously expressed in human gastric cancer (GC) tissues in which its higher expression generally correlated with the poor prognosis of patients. Small interfering RNA (siRNA)-mediated knockdown of TRIM25 expression in MGC-803 and AGS cells had no effects on cell proliferation, whereas reduced cell migration and invasion. Gene set enrichment analysis on The Cancer Genome Atlas stomach adenocarcinoma (STAD) dataset revealed that several signaling pathways, including the migration, E-cadherin and transforming growth factor-β (TGF-β) pathways, were enriched in TRIM25 higher expression patients. Moreover, ectopic expression of TRIM25 in a GC cell line with lower expression of TRIM25 significantly promoted the migration and invasion. Further experiments with TGF-β inhibitor suggested that TRIM25 may exert its function through TGF-β pathway. In summary, our results indicate that TRIM25 acts as an oncogene in GC and thus presents a novel target for the detection and treatment of GC.

TRIM25 blockade by RNA interference inhibited migration and invasion of gastric cancer cells through TGF-β signaling

PubMed Central

Zhu, Zhenya; Wang, Yong; Zhang, Chunhui; Yu, Shiyong; Zhu, Qi; Hou, Kun; Yan, Bo

2016-01-01

Tripartite Motif Containing 25 (TRIM25), a member of TRIM proteins, has been found abnormally expressed in cancers of female reproductive system. Here, TRIM25 was conspicuously expressed in human gastric cancer (GC) tissues in which its higher expression generally correlated with the poor prognosis of patients. Small interfering RNA (siRNA)-mediated knockdown of TRIM25 expression in MGC-803 and AGS cells had no effects on cell proliferation, whereas reduced cell migration and invasion. Gene set enrichment analysis on The Cancer Genome Atlas stomach adenocarcinoma (STAD) dataset revealed that several signaling pathways, including the migration, E-cadherin and transforming growth factor-β (TGF-β) pathways, were enriched in TRIM25 higher expression patients. Moreover, ectopic expression of TRIM25 in a GC cell line with lower expression of TRIM25 significantly promoted the migration and invasion. Further experiments with TGF-β inhibitor suggested that TRIM25 may exert its function through TGF-β pathway. In summary, our results indicate that TRIM25 acts as an oncogene in GC and thus presents a novel target for the detection and treatment of GC. PMID:26754079

Cleavage of cohesin rings coordinates the separation of centrioles and chromatids.

PubMed

Schöckel, Laura; Möckel, Martin; Mayer, Bernd; Boos, Dominik; Stemmann, Olaf

2011-07-10

Cohesin pairs sister chromatids by forming a tripartite Scc1-Smc1-Smc3 ring around them. In mitosis, cohesin is removed from chromosome arms by the phosphorylation-dependent prophase pathway. Centromeric cohesin is protected by shugoshin 1 and protein phosphatase 2A (Sgo1-PP2A) and opened only in anaphase by separase-dependent cleavage of Scc1 (refs 4-6). Following chromosome segregation, centrioles loosen their tight orthogonal arrangement, which licenses later centrosome duplication in S phase. Although a role of separase in centriole disengagement has been reported, the molecular details of this process remain enigmatic. Here, we identify cohesin as a centriole-engagement factor. Both premature sister-chromatid separation and centriole disengagement are induced by ectopic activation of separase or depletion of Sgo1. These unscheduled events are suppressed by expression of non-cleavable Scc1 or inhibition of the prophase pathway. When endogenous Scc1 is replaced by artificially cleavable Scc1, the corresponding site-specific protease triggers centriole disengagement. Separation of centrioles can alternatively be induced by ectopic cleavage of an engineered Smc3. Thus, the chromosome and centrosome cycles exhibit extensive parallels and are coordinated with each other by dual use of the cohesin ring complex.

Brief Reports: Nfix Promotes Survival of Immature Hematopoietic Cells via Regulation of c-Mpl.

PubMed

Hall, Trent; Walker, Megan; Ganuza, Miguel; Holmfeldt, Per; Bordas, Marie; Kang, Guolian; Bi, Wenjian; Palmer, Lance E; Finkelstein, David; McKinney-Freeman, Shannon

2018-02-12

Hematopoietic stem and progenitor cells (HSPCs) are necessary for life-long blood production and replenishment of the hematopoietic system during stress. We recently reported that nuclear factor I/X (Nfix) promotes HSPC survival post-transplant. Here, we report that ectopic expression of Nfix in primary mouse HSPCs extends their ex vivo culture from about 20 to 40 days. HSPCs overexpressing Nfix display hypersensitivity to supportive cytokines and reduced apoptosis when subjected to cytokine deprivation relative to controls. Ectopic Nfix resulted in elevated levels of c-Mpl transcripts and cell surface protein on primary murine HSPCs as well as increased phosphorylation of STAT5, which is known to be activated down-stream of c-MPL. Blocking c-MPL signaling by removal of thrombopoietin or addition of a c-MPL neutralizing antibody negated the antiapoptotic effect of Nfix overexpression on cultured HSPCs. Furthermore, NFIX was capable of binding to and transcriptionally activating a proximal c-Mpl promoter fragment. In sum, these data suggest that NFIX-mediated upregulation of c-Mpl transcription can protect primitive hematopoietic cells from stress ex vivo. Stem Cells 2018. © AlphaMed Press 2018.

RINT-1 interacts with MSP58 within nucleoli and plays a role in ribosomal gene transcription.

PubMed

Yang, Chuan-Pin; Kuo, Yu-Liang; Lee, Yi-Chao; Lee, Kuen-Haur; Chiang, Chi-Wu; Wang, Ju-Ming; Hsu, Che-Chia; Chang, Wen-Chang; Lin, Ding-Yen

2016-09-16

The nucleolus is the cellular site of ribosomal (r)DNA transcription and ribosome biogenesis. The 58-kDa microspherule protein (MSP58) is a nucleolar protein involved in rDNA transcription and cell proliferation. However, regulation of MSP58-mediated rDNA transcription remains unknown. Using a yeast two-hybrid system with MSP58 as bait, we isolated complementary (c)DNA encoding Rad50-interacting protein 1 (RINT-1), as a MSP58-binding protein. RINT-1 was implicated in the cell cycle checkpoint, membrane trafficking, Golgi apparatus and centrosome dynamic integrity, and telomere length control. Both in vitro and in vivo interaction assays showed that MSP58 directly interacts with RINT-1. Interestingly, microscopic studies revealed the co-localization of MSP58, RINT-1, and the upstream binding factor (UBF), a rRNA transcription factor, in the nucleolus. We showed that ectopic expression of MSP58 or RINT-1 resulted in decreased rRNA expression and rDNA promoter activity, whereas knockdown of MSP58 or RINT-1 by siRNA exerted the opposite effect. Coexpression of MSP58 and RINT-1 robustly decreased rRNA synthesis compared to overexpression of either protein alone, whereas depletion of RINT-1 from MSP58-transfected cells enhanced rRNA synthesis. We also found that MSP58, RINT-1, and the UBF were associated with the rDNA promoter using a chromatin immunoprecipitation assay. Because aberrant ribosome biogenesis contributes to neoplastic transformation, our results revealed a novel protein complex involved in the regulation of rRNA gene expression, suggesting a role for MSP58 and RINT-1 in cancer development. Copyright © 2016 Elsevier Inc. All rights reserved.

A proteomic approach reveals transient association of reticulocalbin-3, a novel member of the CREC family, with the precursor of subtilisin-like proprotein convertase, PACE4

PubMed Central

Tsuji, Akihiko; Kikuchi, Yayoi; Sato, Yukimi; Koide, Shizuyo; Yuasa, Keizo; Nagahama, Masami; Matsuda, Yoshiko

2006-01-01

SPCs (subtilisin-like proprotein convertases) are a family of seven structurally related serine endoproteases that are involved in the proteolytic activation of proproteins. In an effort to examine the substrate protein for PACE4 (paired basic amino-acid-cleaving enzyme-4), an SPC, a potent protein inhibitor of PACE4, an α1-antitrypsin RVRR (Arg-Val-Arg-Arg) variant, was expressed in GH4C1 cells. Ectopic expression of the RVRR variant caused accumulation of the 48 kDa protein in cells. Sequence analysis indicates that the 48 kDa protein is a putative Ca2+-binding protein, RCN-3 (reticulocalbin-3), which had previously been predicted by bioinformatic analysis of cDNA from the human hypothalamus. RCN-3 is a member of the CREC (Cab45/reticulocalbin/ERC45/calumenin) family of multiple EF-hand Ca2+-binding proteins localized to the secretory pathway. The most interesting feature of the RCN-3 sequence is the presence of five Arg-Xaa-Xaa-Arg motifs, which represents the target sequence of SPCs. Biosynthetic studies showed that RCN-3 is transiently associated with proPACE4, but not with mature PACE4. Inhibition of PACE4 maturation by a Ca2+ ionophore resulted in accumulation of the proPACE4–RCN-3 complex in cells. Furthermore, autoactivation and secretion of PACE4 was increased upon co-expression with RCN-3. Our findings suggest that selective and transient association of RCN-3 with the precursor of PACE4 plays an important role in the biosynthesis of PACE4. PMID:16433634

Identification of a Novel GJA8 (Cx50) Point Mutation Causes Human Dominant Congenital Cataracts

NASA Astrophysics Data System (ADS)

Ge, Xiang-Lian; Zhang, Yilan; Wu, Yaming; Lv, Jineng; Zhang, Wei; Jin, Zi-Bing; Qu, Jia; Gu, Feng

2014-02-01

Hereditary cataracts are clinically and genetically heterogeneous lens diseases that cause a significant proportion of visual impairment and blindness in children. Human cataracts have been linked with mutations in two genes, GJA3 and GJA8, respectively. To identify the causative mutation in a family with hereditary cataracts, family members were screened for mutations by PCR for both genes. Sequencing the coding regions of GJA8, coding for connexin 50, revealed a C > A transversion at nucleotide 264, which caused p.P88T mutation. To dissect the molecular consequences of this mutation, plasmids carrying wild-type and mutant mouse ORFs of Gja8 were generated and ectopically expressed in HEK293 cells and human lens epithelial cells, respectively. The recombinant proteins were assessed by confocal microscopy and Western blotting. The results demonstrate that the molecular consequences of the p.P88T mutation in GJA8 include changes in connexin 50 protein localization patterns, accumulation of mutant protein, and increased cell growth.

Thymocyte Maturation Is Regulated by the Activity of the Helix-Loop-Helix Protein, E47

PubMed Central

Bain, Gretchen; Quong, Melanie W.; Soloff, Rachel S.; Hedrick, Stephen M.; Murre, Cornelis

1999-01-01

The E2A proteins, E12 and E47, are required for progression through multiple developmental pathways, including early B and T lymphopoiesis. Here, we provide in vitro and in vivo evidence demonstrating that E47 activity regulates double-positive thymocyte maturation. In the absence of E47 activity, positive selection of both major histocompatibility complex (MHC) class I– and class II–restricted T cell receptors (TCRs) is perturbed. Additionally, development of CD8 lineage T cells in an MHC class I–restricted TCR transgenic background is sensitive to the dosage of E47. Mice deficient for E47 display an increase in production of mature CD4 and CD8 lineage T cells. Furthermore, ectopic expression of an E2A inhibitor helix-loop-helix protein, Id3, promotes the in vitro differentiation of an immature T cell line. These results demonstrate that E2A functions as a regulator of thymocyte positive selection. PMID:10587351