GATA4 protects granulosa cell tumors from TRAIL-induced apoptosis

Disturbances in granulosa cell apoptosis have been implicated in the pathogenesis of human granulosa cell tumors (GCTs). Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a potent cytokine that induces apoptosis in a variety of malignancies without toxic effects on benign cells. The aim of this study was to investigate the expression and functionality of the TRAIL receptors DR4 and DR5 in human GCTs. Additionally, we examined the role of GATA4, a transcription factor expressed in normal and malignant granulosa cells, in TRAIL-induced GCT apoptosis. For this purpose, a tissue microarray of 80 primary and 12 recurrent GCTs was subjected to immunohistochemistry for DR4 and DR5, and freshly isolated primary GCT cultures were utilized to evaluate the functional effects of TRAIL on GCT cells. To clarify the role of GATA4 in the regulation of TRAIL-induced apoptosis, a human GCT-derived cell line (KGN) was transduced with lentiviral vectors expressing small hairpin RNAs targeting GATA4 or transfected with adenovirus expressing either wild-type or dominant negative mutant GATA4. We found that receptors DR4 and DR5 are expressed in a vast majority of GCTs as well as in primary GCT cultures, and that TRAIL induces apoptosis in the primary GCT cultures. Moreover, we showed that overexpressing GATA4 protects GCTs from TRAIL-induced apoptosis in vitro, whereas disrupting GATA4 function induces apoptosis and potentiates the apoptotic effect of TRAIL administration. Our results demonstrate that the TRAIL pathway is functional in GCT cells, and suggest that transcription factor GATA4 may function as a survival factor in this ovarian malignancy.

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Tyrosine Phosphorylation of Protein Kinase Cδ Is Essential for Its Apoptotic Effect in Response to Etoposide

Molecular and Cellular Biology ◽

10.1128/mcb.22.1.182-195.2002 ◽

2002 ◽

Vol 22 (1) ◽

pp. 182-195 ◽

Cited By ~ 148

Author(s):

Michal Blass ◽

Ilana Kronfeld ◽

Gila Kazimirsky ◽

Peter M. Blumberg ◽

Chaya Brodie

Keyword(s):

Protein Kinase ◽

Tyrosine Phosphorylation ◽

Dominant Negative ◽

C6 Glioma Cells ◽

Dominant Negative Mutant ◽

Tyrosine Residues ◽

Apoptotic Effect ◽

Induced Apoptosis ◽

Protein Kinase Cδ

ABSTRACT Protein kinase Cδ (PKCδ) is involved in the apoptosis of various cells in response to diverse stimuli. In this study, we characterized the role of PKCδ in the apoptosis of C6 glioma cells in response to etoposide. We found that etoposide induced apoptosis in the C6 cells within 24 to 48 h and arrested the cells in the G1/S phase of the cell cycle. Overexpression of PKCδ increased the apoptotic effect induced by etoposide, whereas the PKCδ selective inhibitor rottlerin and the PKCδ dominant-negative mutant K376R reduced this effect compared to control cells. Etoposide-induced tyrosine phosphorylation of PKCδ and its translocation to the nucleus within 3 h was followed by caspase-dependent cleavage of the enzyme. Using PKC chimeras, we found that both the regulatory and catalytic domains of PKCδ were necessary for its apoptotic effect. The role of tyrosine phosphorylation of PKCδ in the effects of etoposide was examined using cells overexpressing a PKCδ mutant in which five tyrosine residues were mutated to phenylalanine (PKCδ5). These cells exhibited decreased apoptosis in response to etoposide compared to cells overexpressing PKCδ. Likewise, activation of caspase 3 and the cleavage of the PKCδ5 mutant were significantly lower in cells overexpressing PKCδ5. Using mutants of PKCδ altered at individual tyrosine residues, we identified tyrosine 64 and tyrosine 187 as important phosphorylation sites in the apoptotic effect induced by etoposide. Our results suggest a role of PKCδ in the apoptosis induced by etoposide and implicate tyrosine phosphorylation of PKCδ as an important regulator of this effect.

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Mouse Receptor Interacting Protein 3 Does Not Contain a Caspase-Recruiting or a Death Domain but Induces Apoptosis and Activates NF-κB

Molecular and Cellular Biology ◽

10.1128/mcb.19.10.6500 ◽

1999 ◽

Vol 19 (10) ◽

pp. 6500-6508 ◽

Cited By ~ 39

Author(s):

Nanette J. Pazdernik ◽

David B. Donner ◽

Mark G. Goebl ◽

Maureen A. Harrington

Keyword(s):

Sequence Similarity ◽

Kinase Domain ◽

Dominant Negative ◽

Dominant Negative Mutant ◽

Death Domain ◽

Interacting Protein ◽

C Terminus ◽

Catalytically Active ◽

Induced Apoptosis

ABSTRACT The death domain-containing receptor superfamily and their respective downstream mediators control whether or not cells initiate apoptosis or activate NF-κB, events critical for proper immune system function. A screen for upstream activators of NF-κB identified a novel serine-threonine kinase capable of activating NF-κB and inducing apoptosis. Based upon domain organization and sequence similarity, this novel kinase, named mRIP3 (mouse receptor interacting protein 3), appears to be a new RIP family member. RIP, RIP2, and mRIP3 contain an N-terminal kinase domain that share 30 to 40% homology. In contrast to the C-terminal death domain found in RIP or the C-terminal caspase-recruiting domain found in RIP2, the C-terminal tail of mRIP3 contains neither motif and is unique. Despite this feature, overexpression of the mRIP3 C terminus is sufficient to induce apoptosis, suggesting that mRIP3 uses a novel mechanism to induce death. mRIP3 also induced NF-κB activity which was inhibited by overexpression of either dominant-negative NIK or dominant-negative TRAF2. In vitro kinase assays demonstrate that mRIP3 is catalytically active and has autophosphorylation site(s) in the C-terminal domain, but the mRIP3 catalytic activity is not required for mRIP3 induced apoptosis and NF-κB activation. Unlike RIP and RIP2, mRIP3 mRNA is expressed in a subset of adult tissues and is thus likely to be a tissue-specific regulator of apoptosis and NF-κB activity. While the lack of a dominant-negative mutant precludes linking mRIP3 to a known upstream regulator, characterizing the expression pattern and the in vitro functions of mRIP3 provides insight into the mechanism(s) by which cells modulate the balance between survival and death in a cell-type-specific manner.

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Abstract 5278: The Sonic Hedgehog Transcription Factor Gli3 Modulates Ischemia-induced Angiogenesis

Circulation ◽

10.1161/circ.118.suppl_18.s_518-c ◽

2008 ◽

Vol 118 (suppl_18) ◽

Author(s):

Marie-Ange Renault ◽

Jerome Roncalli ◽

Joern Tongers ◽

Sol Misener ◽

Tina Thorne ◽

...

Keyword(s):

Transcription Factor ◽

Limb Ischemia ◽

Capillary Density ◽

Left Ventricular ◽

Dominant Negative ◽

Tube Length ◽

Reduced Ejection Fraction ◽

Border Zones

Gli transcription factors are mediators of hedgehog signaling and have been shown to be critical in several steps during development. We have shown that the Hedgehog pathway is reactivated in the adult cardiovascular system under ischemic conditions, however the specific role of Gli3 has not been elucidated. Adenoviral mediated overexpression of Gli3 promotes HUVEC migration (250±58% of control, p<0.001) while down regulation of Gli3 via siRNA delayed tube formation on Matrigel (total tube length after 8 hours 6.86 vs. 70.76 control), suggesting a possible role of Gli3 in angiogenesis. We next investigated the role of Gli3 in angiogenesis using Gli3 +/− (Gli3 +/XtJ ) mice, a well established model of reduced Gli3 expression. VEGF-induced corneal angiogenesis was impaired in Gli3 +/− mice compared to WT. The role of Gli3 in angiogenesis was then confirmed in two ischemia models. Hind-limb ischemia (HLI) was induced by resection of the left femoral artery. Capillary density was reduced by a mean of 48.40±12.08% in Gli3 +/− mice vs. WT 7, 14 and 28 days. Myocardial infarction (MI) was induced by ligation of the LAD. 28 days after MI, left ventricular function assessed by echo and histological analysis revealed that Gli3 +/− mice exhibit reduced ejection fraction (27.92±4.49% versus 37.56±7.02% for the WT, p=0.004), increased fibrosis area (33.65±9.73% versus 19.81±5.40% for the WT, p=0.007) and a decrease capillary density in the ischemic and border zones. These data indicate that Gli3 deficiency leads to impaired angiogenesis in both ischemic and non ischemic conditions. Moreover, the impairment in ischemia induced neovascularization is associated with more severe impairment of cardiac function after MI. The mechanism of Gli3’s effects was then investigated in vitro . Promoter reporter assays revealed that Gli3 overexpression inhibits Gli-dependent transcription, while Western analysis show increased Akt phosphorylation, activation of the ERK1/2 and increased c-Fos expression. Using a dominant negative Akt expressing virus and a MEK1/2 inhibitor, we show that Gli3 induced-EC migration is dependent on Akt and ERK1/2. These studies provide the first evidence that the Gli3 transcription factor regulates angiogenesis and EC phenotype.

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GSK-3 is an RNA polymerase II phospho-CTD kinase

Nucleic Acids Research ◽

10.1093/nar/gkaa322 ◽

2020 ◽

Vol 48 (11) ◽

pp. 6068-6080 ◽

Cited By ~ 1

Author(s):

Nicolás Nieto Moreno ◽

Florencia Villafañez ◽

Luciana E Giono ◽

Carmen Cuenca ◽

Gastón Soria ◽

...

Keyword(s):

Dna Damage ◽

Rna Polymerase ◽

Rna Polymerase Ii ◽

Glycogen Synthase ◽

Dominant Negative ◽

Dominant Negative Mutant ◽

Transcriptional Elongation ◽

Induced Apoptosis ◽

Carboxy Terminal

Abstract We have previously found that UV-induced DNA damage causes hyperphosphorylation of the carboxy terminal domain (CTD) of RNA polymerase II (RNAPII), inhibition of transcriptional elongation and changes in alternative splicing (AS) due to kinetic coupling between transcription and splicing. In an unbiased search for protein kinases involved in the AS response to DNA damage, we have identified glycogen synthase kinase 3 (GSK-3) as an unforeseen participant. Unlike Cdk9 inhibition, GSK-3 inhibition only prevents CTD hyperphosphorylation triggered by UV but not basal phosphorylation. This effect is not due to differential degradation of the phospho-CTD isoforms and can be reproduced, at the AS level, by overexpression of a kinase-dead GSK-3 dominant negative mutant. GSK-3 inhibition abrogates both the reduction in RNAPII elongation and changes in AS elicited by UV. We show that GSK-3 phosphorylates the CTD in vitro, but preferentially when the substrate is previously phosphorylated, consistently with the requirement of a priming phosphorylation reported for GSK-3 efficacy. In line with a role for GSK-3 in the response to DNA damage, GSK-3 inhibition prevents UV-induced apoptosis. In summary, we uncover a novel role for a widely studied kinase in key steps of eukaryotic transcription and pre-mRNA processing.

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GATA-4 Regulates Bcl-2 Expression in Ovarian Granulosa Cell Tumors

Endocrinology ◽

10.1210/en.2008-0148 ◽

2008 ◽

Vol 149 (11) ◽

pp. 5635-5642 ◽

Cited By ~ 43

Author(s):

Antti Kyrönlahti ◽

Maarit Rämö ◽

Maija Tamminen ◽

Leila Unkila-Kallio ◽

Ralf Butzow ◽

...

Keyword(s):

Cell Fate ◽

Granulosa Cell ◽

Granulosa Cells ◽

Dominant Negative ◽

Rt Pcr ◽

Cyclin D2 ◽

Granulosa Cell Tumors ◽

Ovarian Granulosa Cell ◽

Cell Cycle Regulator Cyclin

Excessive cell proliferation and decreased apoptosis have been implicated in the pathogenesis of ovarian granulosa cell tumors (GCTs). We hypothesized that transcription factor GATA-4 controls expression of the antiapoptotic factor Bcl-2 and the cell cycle regulator cyclin D2 in normal and neoplastic granulosa cells. To test this hypothesis, a tissue microarray based on 80 GCTs was subjected to immunohistochemistry for GATA-4, Bcl-2, and cyclin D2, and the data were correlated to clinical and histopathological parameters. In addition, quantitative RT-PCR for GATA-4, Bcl-2, and cyclin D2 was performed on 21 human GCTs. A mouse GCT model was used to complement these studies. The role of GATA-4 in the regulation of Bcl2 and ccdn2 (coding for cyclin D2) was studied by transactivation assays, and by disrupting GATA-4 function with dominant negative approaches in mouse and human GCT cell lines. We found that GATA-4 expression correlated with Bcl-2 and cyclin D2 expression in human and murine GCTs. Moreover, GATA-4 enhanced Bcl-2 and cyclin D2 promoter activity in murine GCT cells. Whereas GATA-4 overexpression up-regulated and dominant negative GATA-4 suppressed Bcl-2 expression in human GCT cells, the effects on cyclin D2 were negligible. Our results reveal a previously unknown relationship between GATA-4 and Bcl-2 in mammalian granulosa cells and GCTs, and suggest that GATA-4 influences granulosa cell fate by transactivating Bcl-2.

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Calpain-Mediated Bid Cleavage and Calpain-Independent Bak Modulation: Two Separate Pathways in Cisplatin-Induced Apoptosis

Molecular and Cellular Biology ◽

10.1128/mcb.22.9.3003-3013.2002 ◽

2002 ◽

Vol 22 (9) ◽

pp. 3003-3013 ◽

Cited By ~ 163

Author(s):

Aleksandra Mandic ◽

Kristina Viktorsson ◽

Linda Strandberg ◽

Thomas Heiden ◽

Johan Hansson ◽

...

Keyword(s):

Cathepsin L ◽

Inhibitory Effect ◽

Dominant Negative ◽

Dominant Negative Mutant ◽

Drug Induced ◽

Calpain Activation ◽

Isolated Mitochondria ◽

Induced Apoptosis ◽

Calpain Inhibitors

ABSTRACT Calpain is a ubiquitous protease with potential involvement in apoptosis. We report that in human melanoma cells, cisplatin-induced calpain activation occurs early in apoptosis. Calpain activation and subsequent apoptosis were inhibited by calpeptin and PD150606, two calpain inhibitors with different modes of action. Furthermore, cisplatin induced cleavage of the BH3-only protein Bid, yielding a 14-kDa fragment similar to proapoptotic, caspase-cleaved Bid. However, Bid cleavage was inhibited by inhibitors of calpain, but not by inhibitors of caspases or of cathepsin L. Recombinant Bid was cleaved in vitro by both recombinant calpain and by lysates of cisplatin-treated cells. Cleavage was calpeptin sensitive, and the cleavage site was mapped between Gly70 and Arg71. Calpain-cleaved Bid induced cytochrome c release from isolated mitochondria. While calpeptin did not affect cisplatin-induced modulation of Bak to its proapoptotic conformation, a dominant-negative mutant of MEKK1 (dnMEKK) inhibited Bak modulation. dnMEKK did not, however, block Bid cleavage. The combination of dnMEKK and calpeptin had an additive inhibitory effect on apoptosis. In summary, calpain-mediated Bid cleavage is important in drug-induced apoptosis, and cisplatin induces at least two separate apoptotic signaling pathways resulting in Bid cleavage and Bak modulation, respectively.

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Downregulation of transcription factor GATA4 sensitizes human hepatoblastoma cells to doxorubicin-induced apoptosis

Tumor Biology ◽

10.1177/1010428317695016 ◽

2017 ◽

Vol 39 (3) ◽

pp. 101042831769501 ◽

Cited By ~ 3

Author(s):

Tea Soini ◽

Marjut Pihlajoki ◽

Antti Kyrönlahti ◽

Leif C Andersson ◽

David B Wilson ◽

...

Keyword(s):

Transcription Factor ◽

Fetal Liver ◽

Family Members ◽

B Cell Lymphoma ◽

Doxorubicin Treatment ◽

Bcl2 Family ◽

Apoptotic Effect ◽

High Doses ◽

Induced Apoptosis

Hepatoblastoma, the most common type of pediatric liver cancer, is treated with a combination of surgery and chemotherapy. An essential drug in the treatment of hepatoblastoma is doxorubicin, which in high doses is cardiotoxic. This adverse effect is due to downregulation of cardiac expression of transcription factor GATA4, leading in turn to diminished levels of anti-apoptotic BCL2 (B-cell lymphoma 2) protein family members. GATA4 is also expressed in early fetal liver, but absent from normal postnatal hepatocytes. However, GATA4 is highly expressed in hepatoblastoma tissue. In this study, we assessed the role of GATA4 in doxorubicin-induced apoptosis of hepatoblastoma cells. Herein, we demonstrate that doxorubicin decreases GATA4 expression and alters the expression pattern of BCL2 family members, most profoundly that of BCL2 and BAK, in the HUH6 hepatoblastoma cell line. Silencing of GATA4 by siRNA prior to doxorubicin treatment sensitizes HUH6 cells to the apoptotic effect of this drug by further shifting the balance of BCL2 family members to the pro-apoptotic direction. Specifically, expression levels of anti-apoptotic BCL2 were decreased and pro-apoptotic BID were increased after GATA4 silencing. On the whole, our results indicate that since high endogenous levels of transcription factor GATA4 likely protect hepatoblastoma cells from doxorubicin-induced apoptosis, these cells can be rendered more sensitive to the drug by downregulation of GATA4.

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Dominant negative FADD/MORT1 inhibits the development of intestinal intraepithelial lymphocytes with a marked defect on CD8αα+TCRγδ+ T cells

10.1101/313742 ◽

2018 ◽

Author(s):

Xuerui Zhang ◽

Lina Huo ◽

Lulu Song ◽

Zhaoqing Hu ◽

Xinran Wang ◽

...

Keyword(s):

T Cells ◽

Death Receptor ◽

Adaptor Protein ◽

Intraepithelial Lymphocytes ◽

Protective Role ◽

Dominant Negative ◽

Dominant Negative Mutant ◽

Intestinal Intraepithelial Lymphocytes ◽

Induced Apoptosis

AbstractIntestinal intraepithelial lymphocytes are considered to be distinct from thymus-derived cells and are thought to derive locally from cryptopatch (CP) precursors. Although the development and homing of IELs have been studied in some details, the factors controlling their homeostasis are incompletely understood. Here, we demonstrate that FADD, a classic adaptor protein required for death-receptor-induced apoptosis, is a critical regulator of the intestinal IEL development. The mice with a dominant negative mutant of FADD (FADD-DN) display a defective localized intestinal IELs with a marked defect on CD8αα+TCRγδ+ T cells. Since Lin- LPLs have been identified as precursors CP cells for CD8αα+ development, we analyzed lamina propria lymphocytes (LPLs) and found the massive accumulation of IL-7R-lin- LPLs in FADD-DN mice. IL-7 plays a differentiation inducing role in the development of intestinal IELs and its receptor IL-7R is a transcriptional target of Notch1. The level of Notch1 expression also showed very low in Lin- LPLs cells from FADD-DN mice compared with normal mice, indicating a possible molecular mechanism of FADD in the early IEL development. In addition, loss of γδ T-IELs induced by FADD-DN results in a worsening inflammation in murine DSS-induced colitis model, suggesting a protective role of FADD in the intestinal homeostasis.

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Hsc70 is required for endocytosis and clathrin function in Drosophila

The Journal of Cell Biology ◽

10.1083/jcb.200205086 ◽

2002 ◽

Vol 159 (3) ◽

pp. 477-487 ◽

Cited By ~ 79

Author(s):

Henry C. Chang ◽

Sherri L. Newmyer ◽

Michael J. Hull ◽

Melanie Ebersold ◽

Sandra L. Schmid ◽

...

Keyword(s):

Mutant Allele ◽

Imaginal Disc ◽

Substantial Reduction ◽

Dominant Negative ◽

Dominant Negative Mutant ◽

Eye Disc ◽

Mutant Cells ◽

Eye Imaginal Disc

By screening for Drosophila mutants exhibiting aberrant bride of sevenless (Boss) staining patterns on eye imaginal disc epithelia, we have recovered a point mutation in Hsc70-4, the closest homologue to bovine clathrin uncoating ATPase. Although the mutant allele was lethal, analysis of mutant clones generated by FLP/FRT recombination demonstrated that the Sevenless-mediated internalization of Boss was blocked in mutant Hsc70-4 eye disc epithelial cells. Endocytosis of other probes was also greatly inhibited in larval Garland cells. Immunostaining and EM analysis of the mutant cells revealed disruptions in the organization of endosomal/lysosomal compartments, including a substantial reduction in the number of clathrin-coated structures in Garland cells. The Hsc70-4 mutation also interacted genetically with a dominant-negative mutant of dynamin, a gene required for the budding of clathrin-coated vesicles (CCVs). Consistent with these phenotypes, recombinant mutant Hsc70 proteins exhibited diminished clathrin uncoating activity in vitro. Together, these data provide genetic support for the long-suspected role of Hsc70 in clathrin-mediated endocytosis, at least in part by inhibiting the uncoating of CCVs.

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Identification of a hydrogen peroxide-induced PP1-JNK1-Sp1 signaling pathway for gene regulation

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00454.2005 ◽

2006 ◽

Vol 291 (5) ◽

pp. L983-L992 ◽

Cited By ~ 24

Author(s):

Shijian Chu ◽

Thomas J. Ferro

Keyword(s):

Alveolar Epithelial Cell ◽

Dominant Negative ◽

Lung Epithelial Cells ◽

Dominant Negative Mutant ◽

Epithelial Cell Line ◽

Regulation Of Transcription ◽

Jnk Pathway ◽

Human Alveolar Epithelial Cell

Oxidative stress often results in changes in gene expression through the regulation of transcription factors. In this study, we examine how Sp1 phosphorylation is regulated by H2O2 in a human alveolar epithelial cell line (HAE). Treatment of HAE cells with H2O2 increases phosphorylation of Sp1 and activates JNK. To establish a relationship between JNK and Sp1, we show that JNK activator anisomycin increases Sp1 phosphorylation, and JNK inhibitors as well as dominant-negative JNK1 attenuate H2O2-induced Sp1 phosphorylation. Additionally, JNK1 directly phosphorylates Sp1 in vitro, reducing Sp1 binding to DNA. These results demonstrate the role of JNK in H2O2-induced Sp1 phosphorylation. Because H2O2 inhibits Ser/Thr protein phosphatase-1 (PP1), we examined the role of PP1 in the regulation of JNK. Similar to H2O2, inhibition of PP1 induces phosphorylation of Sp1 and activation of JNK in HAE cells. Inhibition of JNK activity using either inhibitors or dominant-negative mutant JNK1 suppresses PP1 inhibition-induced Sp1 phosphorylation. Furthermore, PP1 directly inactivates JNK1 in vitro. These data suggest that 1) H2O2 increases the phosphorylation level of Sp1, 2) Sp1 is a target of the JNK pathway, 3) PP1 regulates JNK activation, and 4) the “PP1-JNK” pathway plays a role in H2O2-induced Sp1 phosphorylation in lung epithelial cells.

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