41 results on '"Musti AM"'
Search Results
2. A novel thyroid transcript negatively regulated by TSH
- Author
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PIANESE L, AVVEDIMENTO VE, D'ESPOSITO F, FELICIELLO A, MONTICELLI A, MUSTI AM, TORTORA G, VARRONE S, COCOZZA, SERGIO, PORCELLINI, ANTONIO, Pianese, Luigi, Porcellini, Antonio, Avvedimento, VITTORIO ENRICO, D'Esposito, Francesco, Feliciello, Antonio, Monticelli, A., Musti, A. M., Tortora, Giampaolo, Varrone, S., Cocozza, Sergio, Pianese, L, Avvedimento, Ve, D'Esposito, F, Feliciello, A, Monticelli, A, Musti, Am, Tortora, G, and Varrone, S
- Published
- 1995
3. Differential phosphorylation of c-Jun and JunD in response to the epidermal growth factor is determined by the structure of MAPK targeting sequences
- Author
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Vinciguerra M, Vivacqua A, Fasanella G, Gallo A, Cuozzo C, Morano A, Maggiolini M, and Musti AM.
- Subjects
integumentary system - Abstract
MAPK phosphorylation of various substrates is mediated by the presence of docking sites, including the D domain and the DEF motif. Depending on the number and sequences of these domains, substrates are phosphorylated by specific subsets of MAPKs. For example, a D domain targets JNK to c-Jun, whereas a DEF motif is required for ERK phosphorylation of c-Fos. JunD, in contrast, contains both D and DEF domains. Here we show that these motifs mediate JunD phosphorylation in response to either ERK or JNK activation. An intact D domain is required for phosphorylation and activation of JunD by both subtypes of MAPK. The DEF motif acts together with the D domain to elicit efficient phosphorylation of JunD in response to the epidermal growth factor (EGF) but has no function on JunD phosphorylation and activation by JNK signaling. Furthermore, we show that conversion of a c-Jun sequence to a canonical DEF domain, as it is present in JunD, elicits c-Jun activation in response to EGF. Our results suggest that evolution of a particular modular system of MAPK targeting sequences has determined a differential response of JunD and c-Jun to ERK activation
- Published
- 2004
4. c-Jun activation is required for 4-hydroxytamoxifen-induced cell death in breast cancer cells
- Author
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Mario Galgani, Antonio Madeo, Anna Maria Musti, Rosamaria Lappano, Marcello Maggiolini, A Gasperi-Campani, Maria Vinciguerra, Madeo, A., Vinciguerra, Maria, Lappano, R., Galgani, Mario, Gasperi Campani, A., Maggiolini, M., Musti, A. M., Madeo A, Vinciguerra M, Lappano R, Galgani M, Gasperi-Campani A, Maggiolini M, and Musti AM
- Subjects
Transcriptional Activation ,Cancer Research ,Programmed cell death ,C-JUN ,C-FOS ,Proto-Oncogene Proteins c-jun ,Breast Neoplasms ,4-HYDROXYTAMOXIFEN ,Biology ,medicine.disease_cause ,Substrate Specificity ,BREAST CANCER ,Cell Line, Tumor ,LNCaP ,Genetics ,medicine ,Animals ,Humans ,Cytotoxic T cell ,Phosphorylation ,Extracellular Signal-Regulated MAP Kinases ,Promoter Regions, Genetic ,skin and connective tissue diseases ,Molecular Biology ,Cell Death ,c-jun ,JNK Mitogen-Activated Protein Kinases ,Cancer ,medicine.disease ,APOPTOSIS ,Gene Expression Regulation, Neoplastic ,Transcription Factor AP-1 ,Tamoxifen ,Receptors, Estrogen ,Organ Specificity ,Apoptosis ,Cancer cell ,Cancer research ,Carcinogenesis ,Proto-Oncogene Proteins c-fos - Abstract
The c-Jun N-terminal kinase (JNK) has been shown to mediate tamoxifen-induced apoptosis in breast cancer cells. However, the downstream mediators of the JNK pathway linking tamoxifen to effectors of apoptosis have yet to be identified. Here we investigated whether c-Jun, the major nuclear target of JNK, plays a role in tamoxifen-induced apoptosis of SkBr3 breast cancer cells. We show that prior to DNA fragmentation and caspase 3/7 activation, cytotoxic concentrations of 4-hydroxytamoxifen (OHT) induced JNK-dependent phosphorylation of c-Jun at JNK sites previously shown to regulate c-Jun mediated apoptosis. Additionally, OHT induced ERK-dependent expression of c-Fos and transactivation of an AP-1-responsive promoter. In particular, the ectopic expression of dominant-negative constructs blocking either AP-1 activity or c-Jun N-terminal phosphorylation prevented DNA fragmentation following OHT treatment. Furthermore, both c-Fos expression and c-Jun N-terminal phosphorylation preceded OHT-dependent activation of caspase 3-7 in different types of tamoxifen-sensitive cancer cells, but not in OHT-resistant LNCaP prostate cancer cells. Taken together, our results indicate that the c-Jun/c-Fos AP-1 complex plays a pro-apoptotic role in OHT-treated cancer cells and suggest that pharmacological boosts of c-Jun activation may be useful in a combination therapy setting to sensitize cancer cells to tamoxifen-mediated cell death.
- Published
- 2009
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5. Multiple members of the mitogen-activated protein kinase family are necessary for PED/PEA-15 anti-apoptotic function
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Anna Perfetti, Umberto Goglia, Angela Cassese, Giovanni Vigliotta, Alessandra Trencia, Stefania Santopietro, Claudia Miele, Francesco Beguinot, Anna Maria Musti, Pietro Formisano, Gerolama Condorelli, Condorelli, Gerolama, Trencia, A, Vigliotta, G, Perfetti, A, Goglia, U, Cassese, A, Musti, Am, Miele, C, Santopietro, S, Formisano, Pietro, and Beguinot, Francesco
- Subjects
MAPK/ERK pathway ,medicine.medical_treatment ,p38 mitogen-activated protein kinases ,Apoptosis ,CHO Cells ,Biology ,p38 Mitogen-Activated Protein Kinases ,Biochemistry ,Cell Line ,chemistry.chemical_compound ,Cricetinae ,medicine ,Animals ,Humans ,Phosphorylation ,cdc42 GTP-Binding Protein ,Molecular Biology ,Anisomycin ,DNA Primers ,Base Sequence ,Kinase ,Growth factor ,MEK inhibitor ,Intracellular Signaling Peptides and Proteins ,JNK Mitogen-Activated Protein Kinases ,Cell Biology ,Phosphoproteins ,Cell biology ,chemistry ,Mitogen-activated protein kinase ,Immunology ,biology.protein ,Mitogen-Activated Protein Kinases ,Apoptosis Regulatory Proteins ,Signal Transduction - Abstract
293 kidney embryonic cells feature very low levels of the anti-apoptotic protein PED. In these cells, expression of PED to levels comparable with those occurring in normal adult cells inhibits apoptosis induced by growth factor deprivation and by exposure to H(2)O(2) or anisomycin. In PED-expressing 293 cells (293(PED)), inhibition of apoptosis upon growth factor deprivation was paralleled by decreased phosphorylation of JNK1/2. In 293(PED) cells, decreased apoptosis induced by anisomycin and H(2)O(2) was also accompanied by block of JNK1/2 and p38 phosphorylations, respectively. Impaired activity of these stress kinases by PED correlated with inhibition of stress-induced Cdc-42, MKK4, and MKK6 activation. At variance with JNK1/2 and p38, PED expression increased basal and growth factor-stimulated Ras-Raf-1 co-precipitation and MAPK phosphorylation and activity. Treatment of 293(PED) cells with the MEK inhibitor PD98059 blocked ERK1/2 phosphorylations with no effect on inhibition of JNK1/2 and p38 activities. Complete rescue of JNK and p38 functions in 293(PED) cells by overexpressing JNK1 or p38, respectively, enabled only partial recovery of apoptotic response to growth factor deprivation and anisomycin. However, simultaneous rescue of JNK and p38 activities accompanied by block of ERK1/2 fully restored these responses. Thus, PED controls activity of the ERK, JNK, and p38 subfamilies of MAPKs. PED anti-apoptotic function in the 293 cells requires PED simultaneous activation of ERK1/2 and inhibition of the JNK/p38 signaling systems by PED.
- Published
- 2002
6. Thyroid transcription factor 1 phosphorylation is not required for protein kinase A-dependent transcription of the thyroglobulin promoter
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Antonio Feliciello, Allevato, G., Musti, A. M., Brasi, D., Gallo, A., Avvedimento, V. E., Gottesman, M. E., Feliciello, Antonio, Allevato, Giovanna, Musti, Am, DE BRASI, Daniele, Gallo, A, Avvedimento, VITTORIO ENRICO, and Gottesman, M. E.
- Subjects
Transcriptional Activation ,Transcription, Genetic ,Thyroid Nuclear Factor 1 ,Transfection ,PC12 Cells ,Thyroglobulin ,TTF1 ,Culture Media, Serum-Free ,Cell Line ,cAMP ,Cyclic AMP ,Animals ,Humans ,PKA ,Phosphorylation ,Promoter Regions, Genetic ,Alanine ,Dose-Response Relationship, Drug ,Nuclear Proteins ,Cyclic AMP-Dependent Protein Kinases ,Precipitin Tests ,Rats ,Enzyme Activation ,COS Cells ,Mutation ,Mutagenesis, Site-Directed ,HeLa Cells ,Plasmids ,Transcription Factors - Abstract
Thyroid transcription factor 1 (TTF1) is a nuclear homeodomain protein that binds to and activates the promoters of several thyroid-specific genes, including that of the thyroglobulin gene (pTg). These genes are also positively regulated by thyroid-stimulating hormone/cyclic AMP (cAMP)/protein kinase A (PKA) signaling. We asked whether PKA directly activates TTF1. We show that cAMP/PKA activates pTg and a synthetic target promoter carrying TTF1 binding site repeats in several cell types. Activation depends on TTF1. Phosphopeptide mapping indicates that TTF1 is constitutively phosphorylated at multiple sites, and that cAMP stimulated phosphorylation of one site, serine 337, in vivo. However, alanine substitution at this residue or at all sites of phosphorylation did not reduce PKA activation of pTg. Thus, PKA stimulates TTF1 transcriptional activity in an indirect manner, perhaps by recruiting to or removing from the target promoter another regulatory factor(s).
7. The Multifaceted Output of c-Jun Biological Activity: Focus at the Junction of CD8 T Cell Activation and Exhaustion.
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Papavassiliou AG and Musti AM
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- Animals, Epigenesis, Genetic, Humans, Neoplasms genetics, Neoplasms immunology, Virus Diseases genetics, Virus Diseases immunology, CD8-Positive T-Lymphocytes immunology, Lymphocyte Activation immunology, Proto-Oncogene Proteins c-jun metabolism
- Abstract
c-Jun is a major component of the dimeric transcription factor activator protein-1 (AP-1), a paradigm for transcriptional response to extracellular signaling, whose components are basic-Leucine Zipper (bZIP) transcription factors of the Jun, Fos, activating transcription factor (ATF), ATF-like (BATF) and Jun dimerization protein 2 (JDP2) gene families. Extracellular signals regulate c-Jun/AP-1 activity at multiple levels, including transcriptional and posttranscriptional regulation of c-Jun expression and transactivity, in turn, establishing the magnitude and the duration of c-Jun/AP-1 activation. Another important level of c-Jun/AP-1 regulation is due to the capability of Jun family members to bind DNA as a heterodimer with every other member of the AP-1 family, and to interact with other classes of transcription factors, thereby acquiring the potential to integrate diverse extrinsic and intrinsic signals into combinatorial regulation of gene expression. Here, we review how these features of c-Jun/AP-1 regulation underlie the multifaceted output of c-Jun biological activity, eliciting quite distinct cellular responses, such as neoplastic transformation, differentiation and apoptosis, in different cell types. In particular, we focus on the current understanding of the role of c-Jun/AP-1 in the response of CD8 T cells to acute infection and cancer. We highlight the transcriptional and epigenetic regulatory mechanisms through which c-Jun/AP-1 participates in the productive immune response of CD8 T cells, and how its downregulation may contribute to the dysfunctional state of tumor infiltrating CD8 T cells. Additionally, we discuss recent insights pointing at c-Jun as a suitable target for immunotherapy-based combination approaches to reinvigorate anti-tumor immune functions.
- Published
- 2020
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8. Crosstalk between Notch, HIF-1α and GPER in Breast Cancer EMT.
- Author
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De Francesco EM, Maggiolini M, and Musti AM
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- Cell Line, Tumor, Disease Progression, Epithelial-Mesenchymal Transition, Female, Gene Expression Regulation, Neoplastic, Humans, Receptor Cross-Talk, Signal Transduction, Breast Neoplasms metabolism, Hypoxia-Inducible Factor 1, alpha Subunit metabolism, Receptors, Estrogen metabolism, Receptors, G-Protein-Coupled metabolism, Receptors, Notch metabolism
- Abstract
The Notch signaling pathway acts in both physiological and pathological conditions, including embryonic development and tumorigenesis. In cancer progression, diverse mechanisms are involved in Notch-mediated biological responses, including angiogenesis and epithelial-mesenchymal-transition (EMT). During EMT, the activation of cellular programs facilitated by transcriptional repressors results in epithelial cells losing their differentiated features, like cell–cell adhesion and apical–basal polarity, whereas they gain motility. As it concerns cancer epithelial cells, EMT may be consequent to the evolution of genetic/epigenetic instability, or triggered by factors that can act within the tumor microenvironment. Following a description of the Notch signaling pathway and its major regulatory nodes, we focus on studies that have given insights into the functional interaction between Notch signaling and either hypoxia or estrogen in breast cancer cells, with a particular focus on EMT. Furthermore, we describe the role of hypoxia signaling in breast cancer cells and discuss recent evidence regarding a functional interaction between HIF-1α and GPER in both breast cancer cells and cancer-associated fibroblasts (CAFs). On the basis of these studies, we propose that a functional network between HIF-1α, GPER and Notch may integrate tumor microenvironmental cues to induce robust EMT in cancer cells. Further investigations are required in order to better understand how hypoxia and estrogen signaling may converge on Notch-mediated EMT within the context of the stroma and tumor cells interaction. However, the data discussed here may anticipate the potential benefits of further pharmacological strategies targeting breast cancer progression.
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- 2018
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9. GPER Mediates Non-Genomic Effects of Estrogen.
- Author
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Pupo M, Maggiolini M, and Musti AM
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- Breast Neoplasms genetics, Breast Neoplasms pathology, Cell Line, Tumor, Cell Migration Assays, Cell Movement drug effects, Chromatin Immunoprecipitation, Connective Tissue Growth Factor genetics, Connective Tissue Growth Factor metabolism, Early Growth Response Protein 1 genetics, Early Growth Response Protein 1 metabolism, Estrogens pharmacology, Female, Fibroblasts metabolism, Fibroblasts pathology, Genes, Reporter, Humans, Luciferases genetics, Luciferases metabolism, Mitogen-Activated Protein Kinases metabolism, Phosphorylation, Promoter Regions, Genetic, Proto-Oncogene Proteins c-fos genetics, Proto-Oncogene Proteins c-fos metabolism, Receptors, Estrogen genetics, Receptors, Estrogen metabolism, Receptors, G-Protein-Coupled genetics, Receptors, G-Protein-Coupled metabolism, Signal Transduction drug effects, Transfection, Workflow, Breast Neoplasms metabolism, Estradiol pharmacology, Fibroblasts drug effects, Receptors, G-Protein-Coupled agonists
- Abstract
Estrogens are important modulators of a broad spectrum of physiological functions in humans. However, despite their beneficial actions, a number of lines of evidence correlate the sustained exposure to exogenous estrogen with increased risk of the onset of various cancers. Mainly these steroid hormones induce their effects by binding and activating estrogen receptors (ERα and ERβ). These receptors belong to the family of ligand-regulated transcription factors, and upon activation they regulate the expression of different target genes by binding directly to specific DNA sequences. On the other hand, in recent years it has become clear that the G protein-coupled estrogen receptor 30 (GPR30/GPER) is able to mediate non-genomic action of estrogens in different cell contexts. In particular, GPER has been shown to specifically bind estrogens, and in turn to functionally cross-react with diverse cell signaling systems such as the epidermal growth factor receptor (EGFR) pathway, the Notch signaling pathway and the mitogen-activated protein kinases (MAPK) pathway. In this chapter we will present some of the different experimental techniques currently used to demonstrate the functional role of GPER in mediating non-genomic actions of estrogens, such as the dual luciferase assay, assessment of the involvement of GPER in the stimulation of cell migration in breast cancer cell lines and in cancer-associated fibroblasts, and chromatin immunoprecipitation assay. Overall, the experimental procedures described herein represent key instruments for assessing the biological role of GPER in mediating non-genomic signals of estrogen.
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- 2016
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10. Oxidative DNA damage induces the ATM-mediated transcriptional suppression of the Wnt inhibitor WIF-1 in systemic sclerosis and fibrosis.
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Svegliati S, Marrone G, Pezone A, Spadoni T, Grieco A, Moroncini G, Grieco D, Vinciguerra M, Agnese S, Jüngel A, Distler O, Musti AM, Gabrielli A, and Avvedimento EV
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- Antibiotics, Antineoplastic chemistry, Biopsy, Bleomycin chemistry, Collagen Type I metabolism, Collagen Type I, alpha 1 Chain, DNA Methylation, Fibroblasts metabolism, Fibrosis, Gene Silencing, Humans, Hydroxamic Acids chemistry, Immunoglobulin G chemistry, Oxygen chemistry, Protein Synthesis Inhibitors chemistry, Reactive Oxygen Species metabolism, Signal Transduction genetics, Adaptor Proteins, Signal Transducing metabolism, Ataxia Telangiectasia Mutated Proteins metabolism, DNA Damage, Oxidative Stress, Repressor Proteins metabolism, Scleroderma, Systemic metabolism, Wnt Proteins metabolism
- Abstract
Systemic sclerosis (SSc) is an autoimmune disease characterized by extensive visceral organ and skin fibrosis. SSc patients have increased production of autoreactive antibodies and Wnt signaling activity. We found that expression of the gene encoding Wnt inhibitor factor 1 (WIF-1) was decreased in fibroblasts from SSc patient biopsies. WIF-1 deficiency in SSc patient cells correlated with increased abundance of the Wnt effector β-catenin and the production of collagen. Knocking down WIF-1 in normal fibroblasts increased Wnt signaling and collagen production. WIF-1 loss and DNA damage were induced in normal fibroblasts by either SSc patient immunoglobulins or oxidative DNA-damaging agents, such as ultraviolet light, hydrogen peroxide, or bleomycin. The DNA damage checkpoint kinase ataxia telangiectasia mutated (ATM) mediated WIF-1 silencing through the phosphorylation of the transcription factor c-Jun, which in turn activated the expression of the gene encoding activating transcription factor 3 (ATF3). ATF3 and c-Jun were recruited together with histone deacetylase 3 (HDAC3) to the WIF-1 promoter and inhibited WIF-1 expression. Preventing the accumulation of reactive oxygen species or inhibiting the activation of ATM, c-Jun, or HDACs restored WIF-1 expression in cultured SSc patient cells. Trichostatin A, an HDAC inhibitor, prevented WIF-1 loss, β-catenin induction, and collagen accumulation in an experimental fibrosis model. Our findings suggest that oxidative DNA damage induced by SSc autoreactive antibodies enables Wnt activation that contributes to fibrosis., (Copyright © 2014, American Association for the Advancement of Science.)
- Published
- 2014
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11. GPER activates Notch signaling in breast cancer cells and cancer-associated fibroblasts (CAFs).
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Pupo M, Pisano A, Abonante S, Maggiolini M, and Musti AM
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- Breast Neoplasms genetics, Cell Line, Tumor, Cyclopentanes pharmacology, Estradiol pharmacology, Female, Fibroblasts drug effects, Fibroblasts metabolism, Fibroblasts pathology, Gene Expression Regulation, Neoplastic, Humans, MCF-7 Cells, Quinolines pharmacology, Receptor Cross-Talk, Signal Transduction drug effects, Up-Regulation drug effects, Breast Neoplasms metabolism, Breast Neoplasms pathology, Receptors, Estrogen metabolism, Receptors, G-Protein-Coupled metabolism, Receptors, Notch metabolism
- Abstract
The G protein-coupled receptor GPR30/GPER has been shown to mediate rapid effects of 17β-estradiol (E2) in diverse types of cancer cells. Here, we provide evidence for a novel crosstalk between GPER and the Notch signaling pathway in breast cancer cells and cancer-associated fibroblasts (CAFs). We show that E2 and the GPER selective ligand G-1 induce both the γ-secretase-dependent activation of Notch-1 and the expression of the Notch target gene Hes-1. These inductions are prevented by knocking down GPER or by using a dominant-negative mutant of the Notch transcriptional co-activator Master-mind like-1 (DN-MAML-1), hence suggesting the involvement of GPER in the Notch-dependent transcription. By performing chromatin-immunoprecipitation experiments and luciferase assays, we also demonstrate that E2 and G-1 induce the recruitment of the intracellular domain of Notch-1 (N1ICD) to the Hes-1 promoter and the transactivation of a Hes-1-reporter gene, respectively. Functionally, the E2 and G-1-induced migration of breast cancer cells and CAFs is abolished in presence of the γ-secretase inhibitor GSI or DN-MAML-1, which both inhibit the Notch signaling pathway. In addition, we demonstrate that E2 and G-1 prevent the expression of VE-Cadherin, while both compounds induce the expression of Snail, a Notch target gene acting as a repressor of cadherins expression. Notably, both GSI and DN-MAML-1 abolish the up-regulation of Snail-1 by E2 and G-1, whereas the use of GSI rescues VE-Cadherin expression. Taken together, our results prove the involvement of the Notch signaling pathway in mediating the effects of estrogenic GPER signaling in breast cancer cells and CAFs., (Copyright © 2013 Elsevier Ltd. All rights reserved.)
- Published
- 2014
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12. Multisite phosphorylation of c-Jun at threonine 91/93/95 triggers the onset of c-Jun pro-apoptotic activity in cerebellar granule neurons.
- Author
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Reddy CE, Albanito L, De Marco P, Aiello D, Maggiolini M, Napoli A, and Musti AM
- Subjects
- Amino Acid Sequence, Animals, HEK293 Cells, Humans, JNK Mitogen-Activated Protein Kinases metabolism, Lithium pharmacology, Mice, Mice, Inbred C57BL, Molecular Sequence Data, Neurites drug effects, Neurites metabolism, Neurons drug effects, PC12 Cells, Phosphopeptides chemistry, Phosphopeptides metabolism, Phosphorylation drug effects, Protein Structure, Tertiary, Proto-Oncogene Proteins c-jun chemistry, Rats, Recombinant Proteins metabolism, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, Apoptosis drug effects, Cerebellum cytology, Neurons cytology, Neurons metabolism, Phosphothreonine metabolism, Proto-Oncogene Proteins c-jun metabolism
- Abstract
Cerebellar granule cell (CGC) apoptosis by trophic/potassium (TK) deprivation is a model of election to study the interplay of pro-apoptotic and pro-survival signaling pathways in neuronal cell death. In this model, the c-Jun N-terminal kinase (JNK) induces pro-apoptotic genes through the c-Jun/activator protein 1 (AP-1) transcription factor. On the other side, a survival pathway initiated by lithium leads to repression of pro-apoptotic c-Jun/AP-1 target genes without interfering with JNK activity. Yet, the mechanism by which lithium inhibits c-Jun activity remains to be elucidated. Here, we used this model system to study the regulation and function of site-specific c-Jun phosphorylation at the S63 and T91/T93 JNK sites in neuronal cell death. We found that TK-deprivation led to c-Jun multiphosphorylation at all three JNK sites. However, immunofluorescence analysis of c-Jun phosphorylation at single cell level revealed that the S63 site was phosphorylated in all c-Jun-expressing cells, whereas the response of T91/T93 phosphorylation was more sensitive, mirroring the switch-like apoptotic response of CGCs. Conversely, lithium prevented T91T93 phosphorylation and cell death without affecting the S63 site, suggesting that T91T93 phosphorylation triggers c-Jun pro-apoptotic activity. Accordingly, a c-Jun mutant lacking the T95 priming site for T91/93 phosphorylation protected CGCs from apoptosis, whereas it was able to induce neurite outgrowth in PC12 cells. Vice versa, a c-Jun mutant bearing aspartate substitution of T95 overwhelmed lithium-mediate protection of CGCs from TK-deprivation, validating that inhibition of T91/T93/T95 phosphorylation underlies the effect of lithium on cell death. Mass spectrometry analysis confirmed multiphosphorylation of c-Jun at T91/T93/T95 in cells. Moreover, JNK phosphorylated recombinant c-Jun at T91/T93 in a T95-dependent manner. On the basis of our results, we propose that T91/T93/T95 multiphosphorylation of c-Jun functions as a sensitivity amplifier of the JNK cascade, setting the threshold for c-Jun pro-apoptotic activity in neuronal cells.
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- 2013
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13. c-Jun is essential for the induction of Il-1β gene expression in in vitro activated Bergmann glial cells.
- Author
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Albanito L, Reddy CE, and Musti AM
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- Animals, Animals, Newborn, Cerebellar Cortex cytology, Cerebellar Cortex growth & development, Cerebellar Cortex metabolism, Coculture Techniques, Mice, Mice, Inbred C57BL, Mice, Knockout, Mice, Transgenic, Neuroglia cytology, Primary Cell Culture, Proto-Oncogene Proteins c-jun deficiency, Proto-Oncogene Proteins c-jun genetics, Gene Expression Regulation, Developmental physiology, Interleukin-1beta biosynthesis, Interleukin-1beta genetics, Neuroglia metabolism, Proto-Oncogene Proteins c-jun physiology
- Abstract
In the central nervous system (CNS), the c-Jun transcription factor has been mainly studied in neuronal cells and coupled to apoptotic and regenerative pathways following brain injury. Besides, several studies have shown a transcriptional role of c-Jun in activated cortical and spinal astrocytes. In contrast, little is known about c-Jun expression and transactivation in Bergmann glial (BG) cells, the radial cerebellar astrocytes playing crucial roles in cerebellar development and physiology. Here, we used neuronal/glial cerebellar cultures from neonatal mice to assess putative functions of c-Jun in BG cells. By performing double immunocytochemical staining of c-Jun and two BG specific markers, S100 and glutamate aspartate transporter (GLAST), we show that c-Jun was highly expressed in radial glial cells derived from Bergmann glia. Bergmann glia-derived cells expressed toll-like receptor 4 and treatment with bacterial lipopolysaccharide (LPS)-induced c-Jun phosphorylation at serine 63, a hallmark of c-Jun transactivation, exclusively in BG cells. Moreover, LPS-induced IL-1β expression and inhibition of c-Jun N-terminal kinase (JNK) activity abolished both c-Jun phosphorylation and the increase of IL-1β mRNA. Notably, LPS failed to induce IL-1β mRNA in neuronal/glial cerebellar cultures generated from conditional knockout mice lacking c-Jun expression in the CNS, indicating the essential role of c-Jun in astroglial-specific induction of IL-1β. Immunohistochemical analyses of c-Jun-expressing cells in the early postnatal cerebellum confirmed in vivo the expression of c-Jun in BG cells and uncovered a dynamic expression of c-Jun during the formation of the BG monolayer. Altogether, our finding underlines a putative role of c-Jun in astroglia-mediated neuroinflammatory dysfunctions of the cerebellum., (Copyright © 2011 Wiley‐Liss, Inc.)
- Published
- 2011
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14. c-Jun activation is required for 4-hydroxytamoxifen-induced cell death in breast cancer cells.
- Author
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Madeo A, Vinciguerra M, Lappano R, Galgani M, Gasperi-Campani A, Maggiolini M, and Musti AM
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- Animals, Breast Neoplasms genetics, Breast Neoplasms metabolism, Cell Death drug effects, Cell Death genetics, Cell Line, Tumor, Extracellular Signal-Regulated MAP Kinases metabolism, Gene Expression Regulation, Neoplastic, Humans, JNK Mitogen-Activated Protein Kinases metabolism, Organ Specificity, Phosphorylation, Promoter Regions, Genetic genetics, Proto-Oncogene Proteins c-fos metabolism, Receptors, Estrogen analysis, Substrate Specificity, Tamoxifen pharmacology, Transcription Factor AP-1 genetics, Transcription Factor AP-1 metabolism, Transcriptional Activation, Breast Neoplasms pathology, Proto-Oncogene Proteins c-jun metabolism, Tamoxifen analogs & derivatives
- Abstract
The c-Jun N-terminal kinase (JNK) has been shown to mediate tamoxifen-induced apoptosis in breast cancer cells. However, the downstream mediators of the JNK pathway linking tamoxifen to effectors of apoptosis have yet to be identified. In this study, we analysed whether c-Jun, the major nuclear target of JNK, has a role in tamoxifen-induced apoptosis of SkBr3 breast cancer cells. We show that before DNA fragmentation and caspase 3/7 activation, cytotoxic concentrations of 4-hydroxytamoxifen (OHT) induced JNK-dependent phosphorylation of c-Jun at JNK sites earlier shown to regulate c-Jun-mediated apoptosis. In addition, OHT induced ERK-dependent expression of c-Fos and transactivation of an AP-1-responsive promoter. In particular, the ectopic expression of dominant-negative constructs blocking either AP-1 activity or c-Jun N-terminal phosphorylation prevented DNA fragmentation after OHT treatment. Furthermore, both c-Fos expression and c-Jun N-terminal phosphorylation preceded OHT-dependent activation of caspase 3-7 in different types of tamoxifen-sensitive cancer cells, but not in OHT-resistant LNCaP prostate cancer cells. Taken together, our results indicate that the c-Jun/c-Fos AP-1 complex has a pro-apoptotic role in OHT-treated cancer cells and suggest that pharmacological boosts of c-Jun activation may be useful in a combination therapy setting to sensitize cancer cells to tamoxifen-mediated cell death.
- Published
- 2010
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15. A cross-talk between the androgen receptor and the epidermal growth factor receptor leads to p38MAPK-dependent activation of mTOR and cyclinD1 expression in prostate and lung cancer cells.
- Author
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Recchia AG, Musti AM, Lanzino M, Panno ML, Turano E, Zumpano R, Belfiore A, Andò S, and Maggiolini M
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- Cell Cycle, Cell Line, Tumor, Cell Proliferation, Cyclin D1 genetics, Gene Expression Regulation, Neoplastic, Humans, Lung Neoplasms pathology, Male, Prostatic Neoplasms pathology, Protein Kinases genetics, Receptor Cross-Talk, Signal Transduction, TOR Serine-Threonine Kinases, Cyclin D1 metabolism, ErbB Receptors metabolism, Lung Neoplasms metabolism, Prostatic Neoplasms metabolism, Protein Kinases metabolism, Receptors, Androgen metabolism, p38 Mitogen-Activated Protein Kinases metabolism
- Abstract
In androgen sensitive LNCaP prostate cancer cells, the proliferation induced by the epidermal growth factor (EGF) involves a cross-talk between the EGF receptor (EGFR) and the androgen receptor (AR). In lung cancer the role of the EGF-EGFR transduction pathway has been documented, whereas androgen activity has received less attention. Here we demonstrate that in LNCaP and A549 non-small cell lung cancer (NSCLC), AR and EGFR are required for either 5alpha-dihydrotestosterone (DHT) or EGF-stimulated cell growth. Only EGF activated ERK signaling and up-regulated early gene expression, while DHT triggered the expression of classical AR-responsive genes with the exception of the EGF-induced PSA transcript in A549 cells. DHT and EGF up-regulated cyclinD1 (CD1) at both mRNA and protein levels in A549 cells, while in LNCaP cells each mitogen increased only CD1 protein expression. In both cell contexts, CD1 up-regulation was prevented by selective inhibitors as well as by knock-down of either AR or EGFR and also inhibiting p38MAPK and the mammalian target of rapamycin (mTOR) pathways. Interestingly, p38MAPK and mTOR repression prevented the activation of the mTOR target ribosomal p70S6 kinase induced by DHT and EGF, indicating that p38MAPK acts as an upstream mTOR regulator. In addition, the proliferative effects promoted by both DHT and EGF in LNCaP and A549 cancer cells were no longer observed blocking either p38MAPK or mTOR activity. Hence, our data suggest that p38MAPK-dependent activation of the mTOR/CD1 pathway may represent a mechanism through which AR and EGFR cross-talk contributes to prostate and lung cancer progression.
- Published
- 2009
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16. Negative charged threonine 95 of c-Jun is essential for c-Jun N-terminal kinase-dependent phosphorylation of threonine 91/93 and stress-induced c-Jun biological activity.
- Author
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Vinciguerra M, Esposito I, Salzano S, Madeo A, Nagel G, Maggiolini M, Gallo A, and Musti AM
- Subjects
- Amino Acid Sequence, Amino Acid Substitution, Anions chemistry, Anions metabolism, Aspartic Acid genetics, Aspartic Acid metabolism, Cells, Cultured, Humans, Molecular Sequence Data, Phosphorylation, Proto-Oncogene Proteins c-jun chemistry, Proto-Oncogene Proteins c-jun genetics, Proto-Oncogene Proteins c-jun physiology, Threonine chemistry, Transcriptional Activation, DNA Damage physiology, JNK Mitogen-Activated Protein Kinases metabolism, Proto-Oncogene Proteins c-jun metabolism, Threonine metabolism, Threonine physiology
- Abstract
Activation of c-Jun, a major component of the AP-1 transcription factor, represents a paradigm for transcriptional response to stress. Transactivation of c-Jun is regulated by Jun-N-terminal kinases (JNKs) through phosphorylation at serine 63 and 73 (S63/S73), as well as at threonine 91 and 93 (T91/T93). How these two groups of phosphoacceptor sites respond to different grades of genotoxic stress and whether DNA-damage pathways influence the extent of their JNK-dependent phosphorylations remain to be elucidated. Here, we show that following a short exposure to the DNA-damaging compound etoposide, c-Jun phosphorylation is restricted to S63/S73. In contrast, JNK-dependent phosphorylation of T91/T93 requires continuous exposure to the drug and is impaired by caffeine treatment or alanine substitution of the adjacent threonine 95 (T95). Conversely, c-Jun mutations switching the T95/Q96 site into a canonical site for mitogen activated protein kinase (MAPK) phosphorylation (T95/P96) rescues T91/T93 phosphorylation in presence of caffeine, suggesting that a preceding phosphorylation at T95 exposes T91/T93 to JNK-dependent phosphorylation. Moreover, we show that alanine substitution at T95 impairs c-Jun transactivation and c-Jun-mediated cell death, indicating that negatively charged T95 is a general constraint for c-Jun activation. Hence, our study suggests that c-Jun may sense the strength of genotoxic stress through DNA-damage dependent phosphorylation of T95, which in turn augments c-Jun transactivation by JNKs.
- Published
- 2008
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17. G protein-coupled receptor 30 (GPR30) mediates gene expression changes and growth response to 17beta-estradiol and selective GPR30 ligand G-1 in ovarian cancer cells.
- Author
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Albanito L, Madeo A, Lappano R, Vivacqua A, Rago V, Carpino A, Oprea TI, Prossnitz ER, Musti AM, Andò S, and Maggiolini M
- Subjects
- Animals, Breast Neoplasms genetics, Breast Neoplasms metabolism, Breast Neoplasms pathology, Cell Growth Processes, Cell Line, Tumor, Estrogen Receptor alpha metabolism, Female, Genes, fos, Humans, Ovarian Neoplasms metabolism, Ovarian Neoplasms pathology, Promoter Regions, Genetic, Proto-Oncogene Proteins c-fos biosynthesis, Proto-Oncogene Proteins c-fos genetics, RNA, Messenger biosynthesis, RNA, Messenger genetics, Receptors, Estrogen, Receptors, G-Protein-Coupled metabolism, Signal Transduction, Cyclopentanes pharmacology, Estradiol pharmacology, Gene Expression Regulation, Neoplastic, Ovarian Neoplasms genetics, Quinolines pharmacology, Receptors, G-Protein-Coupled physiology
- Abstract
Estrogens play a crucial role in the development of ovarian tumors; however, the signal transduction pathways involved in hormone action are still poorly defined. The orphan G protein-coupled receptor 30 (GPR30) mediates the nongenomic signaling of 17beta-estradiol (E2) in a variety of estrogen-sensitive cancer cells through activation of the epidermal growth factor receptor (EGFR) pathway. Whether estrogen receptor alpha (ERalpha) also contributes to GPR30/EGFR signaling is less understood. Here, we show that, in ERalpha-positive BG-1 ovarian cancer cells, both E2 and the GPR30-selective ligand G-1 induced c-fos expression and estrogen-responsive element (ERE)-independent activity of a c-fos reporter gene, whereas only E2 stimulated an ERE-responsive reporter gene, indicating that GPR30 signaling does not activate ERalpha-mediated transcription. Similarly, both ligands up-regulated cyclin D1, cyclin E, and cyclin A, whereas only E2 enhanced progesterone receptor expression. Moreover, both GPR30 and ERalpha expression are required for c-fos stimulation and extracellular signal-regulated kinase (ERK) activation in response to either E2 or G-1. Inhibition of the EGFR transduction pathway inhibited c-fos stimulation and ERK activation by either ligand, suggesting that in ovarian cancer cells GPR30/EGFR signaling relays on ERalpha expression. Interestingly, we show that both GPR30 and ERalpha expression along with active EGFR signaling are required for E2-stimulated and G-1-stimulated proliferation of ovarian cancer cells. Because G-1 was able to induce both c-fos expression and proliferation in the ERalpha-negative/GPR30-positive SKBR3 breast cancer cells, the requirement for ERalpha expression in GPR30/EGFR signaling may depend on the specific cellular context of different tumor types.
- Published
- 2007
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18. 17beta-estradiol, genistein, and 4-hydroxytamoxifen induce the proliferation of thyroid cancer cells through the g protein-coupled receptor GPR30.
- Author
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Vivacqua A, Bonofiglio D, Albanito L, Madeo A, Rago V, Carpino A, Musti AM, Picard D, Andò S, and Maggiolini M
- Subjects
- Antineoplastic Agents pharmacology, Carcinoma metabolism, Cell Line, Tumor, Cell Proliferation, Cyclin A metabolism, Cyclin D1 metabolism, Humans, Promoter Regions, Genetic, Proto-Oncogene Proteins c-fos genetics, Proto-Oncogene Proteins c-fos metabolism, RNA, Messenger metabolism, Receptors, Estrogen metabolism, Signal Transduction, Tamoxifen pharmacology, Thyroid Neoplasms metabolism, Transfection, Carcinoma genetics, Estradiol pharmacology, Gene Expression Regulation, Neoplastic, Genistein pharmacology, Receptors, G-Protein-Coupled metabolism, Tamoxifen analogs & derivatives, Thyroid Neoplasms genetics
- Abstract
The higher incidence of thyroid carcinoma (TC) in women during reproductive years compared with men and the increased risk associated with the therapeutic use of estrogens have suggested a pathogenetic role exerted by these steroids in the development of TC. In the present study, we evaluated the potential of 17beta-estradiol (E2), genistein (G), and 4-hydroxyta-moxifen (OHT) to regulate the expression of diverse estrogen target genes and the proliferation of human WRO, FRO, and ARO thyroid carcinoma cells, which were used as a model system. We have ascertained that ARO cells are devoid of estrogen receptors (ERs), whereas both WRO and FRO cells express a single variant of ERalpha that was neither transactivated, modulated, nor translocated into the nucleus upon treatment with ligands. However, E2, G, and OHT were able either to induce the transcriptional activity of c-fos promoter constructs, including those lacking the estrogen-responsive elements, or to increase c-fos, cyclin A, and D1 expression. It is noteworthy that we have demonstrated that the G protein-coupled receptor 30 (GPR30) and the mitogen-activated protein kinase (MAPK) pathway mediate both the up-regulation of c-fos and the growth response to E2, G, and OHT in TC cells studied, because these stimulatory effects were prevented by silencing GPR30 and using the MEK inhibitor 2'-amino-3'-methoxyflavone (PD 98059). Our findings provide new insight into the molecular mechanisms through which estrogens may induce the progression of TC.
- Published
- 2006
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19. The G protein-coupled receptor GPR30 mediates the proliferative effects induced by 17beta-estradiol and hydroxytamoxifen in endometrial cancer cells.
- Author
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Vivacqua A, Bonofiglio D, Recchia AG, Musti AM, Picard D, Andò S, and Maggiolini M
- Subjects
- Androstadienes pharmacology, Cell Proliferation drug effects, Endometrial Neoplasms drug therapy, Endometrial Neoplasms metabolism, Enzyme Activation drug effects, Enzyme Inhibitors pharmacology, Estrogen Receptor alpha drug effects, Estrogen Receptor alpha metabolism, Female, Flavonoids pharmacology, Gene Expression Regulation, Neoplastic drug effects, Humans, MAP Kinase Signaling System drug effects, Phosphatidylinositol 3-Kinases metabolism, Phosphoinositide-3 Kinase Inhibitors, Promoter Regions, Genetic, Protein Splicing, Proto-Oncogene Proteins c-fos drug effects, Proto-Oncogene Proteins c-fos genetics, Proto-Oncogene Proteins c-fos metabolism, Receptors, Estrogen, Receptors, G-Protein-Coupled drug effects, Tamoxifen pharmacology, Transcriptional Activation, Tumor Cells, Cultured, Up-Regulation, Wortmannin, Endometrial Neoplasms pathology, Estradiol pharmacology, Receptors, G-Protein-Coupled metabolism, Selective Estrogen Receptor Modulators pharmacology, Tamoxifen analogs & derivatives
- Abstract
The growth of both normal and transformed epithelial cells of the female reproductive system is stimulated by estrogens, mainly through the activation of estrogen receptor alpha (ERalpha), which is a ligand-regulated transcription factor. The selective ER modulator tamoxifen (TAM) has been widely used as an ER antagonist in breast tumor; however, long-term treatment is associated with an increased risk of endometrial cancer. To provide new insights into the potential mechanisms involved in the agonistic activity exerted by TAM in the uterus, we evaluated the potential of 4-hydroxytamoxifen (OHT), the active metabolite of TAM, to transactivate wild-type ERalpha and its splice variant expressed in Ishikawa and HEC1A endometrial tumor cells, respectively. OHT was able to antagonize only the activation of ERalpha by 17beta-estradiol (E2) in Ishikawa cells, whereas it up-regulated c-fos expression in a rapid manner similar to E2 and independently of ERalpha in both cell lines. This stimulation occurred through the G protein-coupled receptor named GPR30 and required Src-related and epidermal growth factor receptor tyrosine kinase activities, along with the activation of both ERK1/2 and phosphatidylinositol 3-kinase/AKT pathways. Most importantly, OHT, like E2, stimulated the proliferation of Ishikawa as well as HEC1A cells. Transfecting a GPR30 antisense expression vector in both endometrial cancer cell lines, OHT was no longer able to induce growth effects, whereas the proliferative response to E2 was completely abrogated only in HEC1A cells. Furthermore, in the presence of the inhibitors of MAPK and phosphatidylinositol 3-kinase pathways, PD 98059 and wortmannin, respectively, E2 and OHT did not elicit growth stimulation. Our data demonstrate a new mode of action of E2 and OHT in endometrial cancer cells, contributing to a better understanding of the molecular mechanisms involved in their uterine agonistic activity.
- Published
- 2006
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20. The G protein-coupled receptor GPR30 mediates c-fos up-regulation by 17beta-estradiol and phytoestrogens in breast cancer cells.
- Author
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Maggiolini M, Vivacqua A, Fasanella G, Recchia AG, Sisci D, Pezzi V, Montanaro D, Musti AM, Picard D, and Andò S
- Subjects
- Breast Neoplasms genetics, Cell Line, Tumor, Estrogen Receptor alpha, Genistein pharmacology, Humans, Mitogen-Activated Protein Kinase 1 metabolism, Mitogen-Activated Protein Kinase 3, Mitogen-Activated Protein Kinases metabolism, Oligoribonucleotides, Antisense pharmacology, Phosphorylation, Phytoestrogens, Plasmids genetics, Proto-Oncogene Mas, Proto-Oncogene Proteins c-fos antagonists & inhibitors, Proto-Oncogene Proteins c-fos genetics, Quercetin pharmacology, RNA, Messenger biosynthesis, Receptors, Estrogen metabolism, Receptors, G-Protein-Coupled genetics, Signal Transduction, Transcriptional Activation drug effects, Transfection, Up-Regulation drug effects, Breast Neoplasms metabolism, Estradiol pharmacology, Isoflavones pharmacology, Plant Preparations pharmacology, Proto-Oncogene Proteins c-fos biosynthesis, Receptors, G-Protein-Coupled physiology
- Abstract
A growing body of evidence concerning estrogen effects cannot be explained by the classic model of hormone action, which involves the binding to estrogen receptors (ERs) alpha and ERbeta and the interaction of the steroid-receptor complex with specific DNA sequences associated with target genes. Using c-fos proto-oncogene expression as an early molecular sensor of estrogen action in ERalpha-positive MCF7 and ER-negative SKBR3 breast cancer cells, we have discovered that 17beta-estradiol (E2), and the two major phytoestrogens, genistein and quercetin, stimulate c-fos expression through ERalpha as well as through an ER-independent manner via the G protein-coupled receptor homologue GPR30. The c-fos response is repressed in GPR30-expressing SKBR3 cells transfected with an antisense oligonucleotide against GPR30 and reconstituted in GPR30-deficient MDA-MB 231 and BT-20 breast cancer cells transfected with a GPR30 expression vector. GPR30-dependent activation of ERK1/2 by E2 and phytoestrogens occurs via a Gbetagamma-associated pertussis toxin-sensitive pathway that requires both Src-related and EGF receptor tyrosine kinase activities. The ability of E2 and phytoestrogens to regulate the expression of growth-related genes such as c-fos even in the absence of ER has interesting implications for understanding breast cancer progression.
- Published
- 2004
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21. Menin uncouples Elk-1, JunD and c-Jun phosphorylation from MAP kinase activation.
- Author
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Gallo A, Cuozzo C, Esposito I, Maggiolini M, Bonofiglio D, Vivacqua A, Garramone M, Weiss C, Bohmann D, and Musti AM
- Subjects
- Animals, Binding Sites, Chloramphenicol O-Acetyltransferase metabolism, Down-Regulation, Glutathione Transferase, HeLa Cells, Humans, Immunoblotting, MAP Kinase Kinase 4, Mitogen-Activated Protein Kinase Kinases pharmacology, Mitogen-Activated Protein Kinases pharmacology, Phosphorylation, Plasmids, Promoter Regions, Genetic, Protein Serine-Threonine Kinases pharmacology, Proto-Oncogene Proteins genetics, Proto-Oncogene Proteins c-fos metabolism, Proto-Oncogene Proteins c-jun genetics, Transcription Factors genetics, Transcription Factors metabolism, Transcriptional Activation, ets-Domain Protein Elk-1, Calcium-Calmodulin-Dependent Protein Kinases metabolism, DNA-Binding Proteins, JNK Mitogen-Activated Protein Kinases, MAP Kinase Kinase Kinase 1, Neoplasm Proteins pharmacology, Proto-Oncogene Proteins metabolism, Proto-Oncogene Proteins c-jun metabolism, Transcription Factor AP-1 metabolism
- Abstract
Menin, a nuclear protein encoded by the tumor suppressor gene MEN1, interacts with the AP-1 transcription factor JunD and inhibits its transcriptional activity. In addition, overexpression of Menin counteracts Ras-induced tumorigenesis. We show that Menin inhibits ERK-dependent phosphorylation and activation of both JunD and the Ets-domain transcription factor Elk-1. We also show that Menin represses the inducible activity of the c-fos promoter. Furthermore, Menin expression inhibits Jun N-terminal kinase (JNK)-mediated phosphorylation of both JunD and c-Jun. Kinase assays show that Menin overexpression does not interfere with activation of either ERK2 or JNK1, suggesting that Menin acts at a level downstream of MAPK activation. An N-terminal deletion mutant of Menin that cannot inhibit JunD phosphorylation by JNK, can still repress JunD phosphorylation by ERK2, suggesting that Menin interferes with ERK and JNK pathways through two distinct inhibitory mechanisms. Taken together, our data suggest that Menin uncouples ERK and JNK activation from phosphorylation of their nuclear targets Elk-1, JunD and c-Jun, hence inhibiting accumulation of active Fos/Jun heterodimers. This study provides new molecular insights into the tumor suppressor function of Menin and suggests a mechanism by which Menin may interfere with Ras-dependent cell transformation and oncogenesis.
- Published
- 2002
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22. Channelrhodopsin-1: a light-gated proton channel in green algae.
- Author
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Nagel G, Ollig D, Fuhrmann M, Kateriya S, Musti AM, Bamberg E, and Hegemann P
- Subjects
- Amino Acid Sequence, Animals, Bacteriorhodopsins chemistry, Bacteriorhodopsins metabolism, Butyric Acid pharmacology, Chlamydomonas reinhardtii chemistry, Chlamydomonas reinhardtii genetics, Electric Conductivity, Hydrogen-Ion Concentration, Ion Channel Gating, Ion Channels genetics, Ion Transport, Membrane Potentials, Molecular Sequence Data, Oocytes, Patch-Clamp Techniques, RNA, Complementary, Recombinant Proteins metabolism, Retinaldehyde pharmacology, Sequence Alignment, Temperature, Xenopus laevis, Chlamydomonas reinhardtii metabolism, Ion Channels chemistry, Ion Channels metabolism, Light, Protons
- Abstract
Phototaxis and photophobic responses of green algae are mediated by rhodopsins with microbial-type chromophores. We report a complementary DNA sequence in the green alga Chlamydomonas reinhardtii that encodes a microbial opsin-related protein, which we term Channelopsin-1. The hydrophobic core region of the protein shows homology to the light-activated proton pump bacteriorhodopsin. Expression of Channelopsin-1, or only the hydrophobic core, in Xenopus laevis oocytes in the presence of all-trans retinal produces a light-gated conductance that shows characteristics of a channel selectively permeable for protons. We suggest that Channelrhodopsins are involved in phototaxis of green algae.
- Published
- 2002
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23. Multiple members of the mitogen-activated protein kinase family are necessary for PED/PEA-15 anti-apoptotic function.
- Author
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Condorelli G, Trencia A, Vigliotta G, Perfetti A, Goglia U, Cassese A, Musti AM, Miele C, Santopietro S, Formisano P, and Beguinot F
- Subjects
- Animals, Apoptosis Regulatory Proteins, Base Sequence, CHO Cells, Cell Line, Cricetinae, DNA Primers, Humans, Intracellular Signaling Peptides and Proteins, JNK Mitogen-Activated Protein Kinases, Mitogen-Activated Protein Kinases antagonists & inhibitors, Phosphorylation, Signal Transduction, cdc42 GTP-Binding Protein metabolism, p38 Mitogen-Activated Protein Kinases, Apoptosis physiology, Mitogen-Activated Protein Kinases metabolism, Phosphoproteins physiology
- Abstract
293 kidney embryonic cells feature very low levels of the anti-apoptotic protein PED. In these cells, expression of PED to levels comparable with those occurring in normal adult cells inhibits apoptosis induced by growth factor deprivation and by exposure to H(2)O(2) or anisomycin. In PED-expressing 293 cells (293(PED)), inhibition of apoptosis upon growth factor deprivation was paralleled by decreased phosphorylation of JNK1/2. In 293(PED) cells, decreased apoptosis induced by anisomycin and H(2)O(2) was also accompanied by block of JNK1/2 and p38 phosphorylations, respectively. Impaired activity of these stress kinases by PED correlated with inhibition of stress-induced Cdc-42, MKK4, and MKK6 activation. At variance with JNK1/2 and p38, PED expression increased basal and growth factor-stimulated Ras-Raf-1 co-precipitation and MAPK phosphorylation and activity. Treatment of 293(PED) cells with the MEK inhibitor PD98059 blocked ERK1/2 phosphorylations with no effect on inhibition of JNK1/2 and p38 activities. Complete rescue of JNK and p38 functions in 293(PED) cells by overexpressing JNK1 or p38, respectively, enabled only partial recovery of apoptotic response to growth factor deprivation and anisomycin. However, simultaneous rescue of JNK and p38 activities accompanied by block of ERK1/2 fully restored these responses. Thus, PED controls activity of the ERK, JNK, and p38 subfamilies of MAPKs. PED anti-apoptotic function in the 293 cells requires PED simultaneous activation of ERK1/2 and inhibition of the JNK/p38 signaling systems by PED.
- Published
- 2002
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24. The DEXD/H-box RNA helicase RHII/Gu is a co-factor for c-Jun-activated transcription.
- Author
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Westermarck J, Weiss C, Saffrich R, Kast J, Musti AM, Wessely M, Ansorge W, Séraphin B, Wilm M, Valdez BC, and Bohmann D
- Subjects
- Animals, Cell Differentiation genetics, DEAD-box RNA Helicases, Humans, PC12 Cells, Protein Binding, Proto-Oncogene Proteins c-jun metabolism, RNA Helicases metabolism, Rats, Transcriptional Activation, Proto-Oncogene Proteins c-jun genetics, RNA Helicases genetics, Transcription, Genetic
- Abstract
Tandem affinity purification (TAP) and mass spectrometric peptide sequencing showed that the DEAD-box RNA helicase RHII/Gu is a functional interaction partner of c-Jun in human cells. The N-terminal transcription activation region of, c-Jun interacts with a C-terminal domain of RHII/Gu. This interaction is stimulated by anisomycin treatment in a manner that is concurrent with, but independent of, c-Jun phosphorylation. A possible explanation for this effect is provided by the observation that RHII/Gu translocates from nucleolus to nucleoplasm upon anisomycin or UV treatment or when JNK signaling is activated by overexpression of a constitutively active form of MEKK1 kinase. Several experiments show that the RNA helicase activity of RHII/Gu supports c-Jun-mediated target gene activation: dominant-negative forms of RHII/Gu, as well as a neutralizing antibody against the enzyme, significantly interfered with c-Jun target gene activity but not with transcription in general. These findings clarify the mechanism of c-Jun-mediated transcriptional regulation, and provide evidence for an involvement of RHII/Gu in stress response and in RNA polymerase II-catalyzed transcription in mammalian cells.
- Published
- 2002
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25. Thyroid transcription factor 1 phosphorylation is not required for protein kinase A-dependent transcription of the thyroglobulin promoter.
- Author
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Feliciello A, Allevato G, Musti AM, De Brasi D, Gallo A, Avvedimento VE, and Gottesman ME
- Subjects
- Alanine chemistry, Animals, COS Cells, Cell Line, Culture Media, Serum-Free, Cyclic AMP metabolism, Dose-Response Relationship, Drug, Enzyme Activation, HeLa Cells, Humans, Mutagenesis, Site-Directed, Mutation, PC12 Cells, Phosphorylation, Plasmids metabolism, Precipitin Tests, Rats, Thyroid Nuclear Factor 1, Transcriptional Activation, Transfection, Cyclic AMP-Dependent Protein Kinases metabolism, Nuclear Proteins metabolism, Promoter Regions, Genetic, Thyroglobulin genetics, Transcription Factors metabolism, Transcription, Genetic
- Abstract
Thyroid transcription factor 1 (TTF1) is a nuclear homeodomain protein that binds to and activates the promoters of several thyroid-specific genes, including that of the thyroglobulin gene (pTg). These genes are also positively regulated by thyroid-stimulating hormone/cyclic AMP (cAMP)/protein kinase A (PKA) signaling. We asked whether PKA directly activates TTF1. We show that cAMP/PKA activates pTg and a synthetic target promoter carrying TTF1 binding site repeats in several cell types. Activation depends on TTF1. Phosphopeptide mapping indicates that TTF1 is constitutively phosphorylated at multiple sites, and that cAMP stimulated phosphorylation of one site, serine 337, in vivo. However, alanine substitution at this residue or at all sites of phosphorylation did not reduce PKA activation of pTg. Thus, PKA stimulates TTF1 transcriptional activity in an indirect manner, perhaps by recruiting to or removing from the target promoter another regulatory factor(s).
- Published
- 2000
26. Determination of functional domains of the human transcription factor PAX8 responsible for its nuclear localization and transactivating potential.
- Author
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Poleev A, Okladnova O, Musti AM, Schneider S, Royer-Pokora B, and Plachov D
- Subjects
- Animals, Cell Line, Cyclic AMP metabolism, DNA-Binding Proteins chemistry, DNA-Binding Proteins genetics, Gene Expression Regulation, Developmental, Genes, Wilms Tumor, Humans, PAX8 Transcription Factor, Paired Box Transcription Factors, Phosphorylation, Promoter Regions, Genetic, Rats, Recombinant Fusion Proteins genetics, Recombinant Fusion Proteins metabolism, Thyroid Gland cytology, Thyroid Gland metabolism, Trans-Activators chemistry, Trans-Activators genetics, Transcription Factors genetics, Tumor Cells, Cultured, Wilms Tumor genetics, Cell Nucleus metabolism, DNA-Binding Proteins metabolism, Nuclear Proteins, Saccharomyces cerevisiae Proteins, Trans-Activators metabolism, Transcriptional Activation
- Abstract
The conserved structure of the transcription factors of the Pax gene family may reflect functional conservation. We have demonstrated that the human Pax8 transcription factor is organized in several functional domains and contains two regions responsible for its nuclear localization, in addition to an activating region at the carboxy terminus of the protein and an inhibitory region encoded by the exon 9 present only in a splice variant PAX8a. Regions of PAX8 determining the nuclear localization of the PAX8A/lacZ fusions contain short amino acid sequences similar to several described nuclear localization sites (NLS). These NLS were identified in the paired domain and between the octapeptide and the residual homeodomain, respectively. The activating domain is encoded by the exons 10 and 11 and its function is modulated by the adjacent domains encoded by the exons 9 and 12. The domain encoded by exon 9 significantly inhibits the function of the activating domain. Pax8 is expressed in thyroid cells and its product binds promoters of the thyroglobulin and thyroperoxidase genes through its paired domain. Thyroid cell growth and differentiation depend on thyrotropin which, by stimulating cAMP synthesis, activates the cAMP-dependent protein kinase A (PKA). We have investigated a link between thyrotropin stimulation and gene activation by Pax8. Stimulation of cAMP synthesis augments Pax8-specific transcription in thyroid cells, indicating that PKA is involved in Pax8 activation. Cotransfection of GAL4/PAX8 fusions and the catalytic subunit of PKA in A126, a PKA-deficient derivative of the PC12 pheochromocytoma cell line, synergistically activates the GAL4-specific reporter, suggesting the activating domain of PAX8 is dependent upon the catalytic subunit of the PKA. We propose that this dependence is due to a hypothetical adaptor which forms a target for PKA and interacts with the activating domain of PAX8. We show that PAX8 isolated from the thyroid cell line FTRL5 is a phosphoprotein in which phosphorylation is not dependant on cAMP pathway activation. Our results suggest that Pax8 is part of the cAMP signaling pathway and mediates thyrotropin-dependent gene activation in thyroid cells. Investigation of the PAX8 expression in a panel of Wilms' tumors shows a striking correlation between the expression of PAX8 and another transcription factor, WT1, indicating that these two genes may interact in vivo.
- Published
- 1997
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27. Reduced ubiquitin-dependent degradation of c-Jun after phosphorylation by MAP kinases.
- Author
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Musti AM, Treier M, and Bohmann D
- Subjects
- 3T3 Cells, Animals, Cell Cycle Proteins metabolism, GTP-Binding Proteins metabolism, Gene Expression Regulation, JNK Mitogen-Activated Protein Kinases, Mice, Phosphorylation, Signal Transduction, Transfection, cdc42 GTP-Binding Protein, Saccharomyces cerevisiae, Calcium-Calmodulin-Dependent Protein Kinases metabolism, Mitogen-Activated Protein Kinases, Proto-Oncogene Proteins c-jun metabolism, Ubiquitins metabolism
- Abstract
The proto-oncogene-encoded transcription factor c-Jun activates genes in response to a number of inducers that act through mitogen-activated protein kinase (MAPK) signal transduction pathways. The activation of c-Jun after phosphorylation by MAPK is accompanied by a reduction in c-Jun ubiquitination and consequent stabilization of the protein. These results illustrate the relevance of regulated protein degradation in the signal-dependent control of gene expression.
- Published
- 1997
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28. The v-Ki-Ras oncogene alters cAMP nuclear signaling by regulating the location and the expression of cAMP-dependent protein kinase IIbeta.
- Author
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Feliciello A, Giuliano P, Porcellini A, Garbi C, Obici S, Mele E, Angotti E, Grieco D, Amabile G, Cassano S, Li Y, Musti AM, Rubin CS, Gottesman ME, and Avvedimento EV
- Subjects
- A Kinase Anchor Proteins, Animals, Blotting, Western, Carrier Proteins, Cell Line, Cell Transformation, Neoplastic, Cyclic AMP pharmacology, Cyclic AMP-Dependent Protein Kinase RIIbeta Subunit, Cyclic AMP-Dependent Protein Kinase Type II, Cyclic AMP-Dependent Protein Kinases biosynthesis, DNA Primers, Gene Expression Regulation, Enzymologic, Mannosidases biosynthesis, Oncogene Protein p21(ras) biosynthesis, Polymerase Chain Reaction, Protein Biosynthesis, Proteins metabolism, Rats, Receptors, Thyrotropin biosynthesis, Recombinant Proteins biosynthesis, Recombinant Proteins metabolism, Sequence Deletion, Thyroglobulin biosynthesis, Transfection, alpha-Mannosidase, Adaptor Proteins, Signal Transducing, Cyclic AMP-Dependent Protein Kinases metabolism, Genes, ras, Signal Transduction
- Abstract
The v-Ki-Ras oncoprotein dedifferentiates thyroid cells and inhibits nuclear accumulation of the catalytic subunit of cAMP-dependent protein kinase. After activation of v-Ras or protein kinase C, the regulatory subunit of type II protein kinase A, RIIbeta, translocates from the membranes to the cytosol. RIIbeta mRNA and protein were eventually depleted. These effects were mimicked by expressing AKAP45, a truncated version of the RII anchor protein, AKAP75. Because AKAP45 lacks membrane targeting domains, it induces the translocation of PKAII to the cytoplasm. Expression of AKAP45 markedly decreased thyroglobulin mRNA levels and inhibited accumulation of C-PKA in the nucleus. Our results suggest that: 1) The localization of PKAII influences cAMP signaling to the nucleus; 2) Ras alters the localization and the expression of PKAII; 3) Translocation of PKAII to the cytoplasm reduces nuclear C-PKA accumulation, resulting in decreased expression of cAMP-dependent genes, including RIIbeta, TSH receptor, and thyroglobulin. The loss of RIIbeta permanently down-regulates thyroid-specific gene expression.
- Published
- 1996
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29. Differential regulation of c-Jun and JunD by ubiquitin-dependent protein degradation.
- Author
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Musti AM, Treier M, Peverali FA, and Bohmann D
- Subjects
- 3T3 Cells, Amino Acid Sequence, Animals, HeLa Cells, Humans, Hydrolysis, Mice, Molecular Sequence Data, Proto-Oncogene Proteins c-jun metabolism, Recombinant Fusion Proteins genetics, Recombinant Fusion Proteins metabolism, Gene Expression Regulation, Proto-Oncogene Proteins c-jun genetics, Ubiquitins metabolism
- Abstract
c-Jun and JunD are two closely related members of the Jun family of transcription factors which markedly differ in their biological functions. Whereas c-Jun behaves as a positive regulator of cell growth and may cause cell transformation when overexpressed, JunD antagonizes both of these effects. To better understand how the activities of c-Jun and JunD are controlled, we investigated how their stabilities within the cell are determined. We show that, in contrast to c-Jun which is degraded following multi ubiquitination, JunD is not efficiently ubiquitinated and exhibits a correspondingly longer half-life. Mutational analysis reveals that the determinant for the difference in ubiquitination resides in the NH2-terminal regions of the proteins which in c-Jun contains the delta-domain.
- Published
- 1996
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30. Ubiquitin in signal transduction and cell transformation.
- Author
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Isaksson A, Musti AM, and Bohmann D
- Subjects
- Animals, Humans, Cell Transformation, Neoplastic, Signal Transduction physiology, Ubiquitins physiology
- Abstract
Since the discovery of ubiquitin-dependent protein degradation almost two decades ago, great strides have been made towards a detailed understanding of the biochemistry of this process (reviewed in [1-3]). It was, however, only in recent years that the physiological role of the ubiquitin system in signal transduction and the regulation of several cell functions started to be appreciated and experimentally addressed. As with other principal mechanisms of signal transduction, such as phosphorylation or GTP hydrolysis, much of the information regarding the role of the ubiquitin system as a component of cell regulation and signaling cascades, was gained in studies of transformation and the control of cell growth. It seems, however, that ubiquitin-dependent proteolysis, and possibly other processes that are controlled by protein ubiquitination, play a role in many aspects of cellular function from the control of differentiation to intracellular trafficking [1,3,4]. Here we will review some of the results that implicate ubiquitin-dependent proteolysis in the control of cell growth and that indicate how perturbations of ubiquitin-dependent degradation of oncogene and tumor suppressor gene products may contribute to cell transformation and oncogenesis.
- Published
- 1996
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31. v-ras and protein kinase C dedifferentiate thyroid cells by down-regulating nuclear cAMP-dependent protein kinase A.
- Author
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Gallo A, Benusiglio E, Bonapace IM, Feliciello A, Cassano S, Garbi C, Musti AM, Gottesman ME, and Avvedimento EV
- Subjects
- Animals, Base Sequence, Cell Differentiation drug effects, Cell Line, Cell Nucleus enzymology, Cyclic AMP pharmacology, Down-Regulation drug effects, Down-Regulation genetics, Fluorescent Antibody Technique, Molecular Sequence Data, Oncogene Protein p21(ras) pharmacology, Protein Kinases metabolism, Rats, Sphingosine pharmacology, Temperature, Thyroid Gland enzymology, Cell Differentiation genetics, Oncogene Protein p21(ras) genetics, Protein Kinase C metabolism, Protein Kinases genetics, Thyroid Gland cytology
- Abstract
Ras proteins are membrane-associated transducers of eternal stimuli to unknown intracellular targets. The constitutively activated v-ras oncogene induces dedifferentiation in thyroid cells. v-Ras appears to act by stimulating protein kinase C (PKC), which inhibits the nuclear migration of the catalytic subunit of the cAMP-dependent protein kinase A (PKA). Nuclear tissue-specific and housekeeping trans-acting factors that are dependent on phosphorylation by PKA are thus inactivated. Exclusion of the PKA subunit from the nucleus could represent a general mechanism for the pleiotropic effects of Ras and PKC on cellular growth and differentiation.
- Published
- 1992
- Full Text
- View/download PDF
32. Reversible inhibition of a thyroid-specific trans-acting factor by Ras.
- Author
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Avvedimento VE, Musti AM, Ueffing M, Obici S, Gallo A, Sanchez M, DeBrasi D, and Gottesman ME
- Subjects
- Animals, Base Sequence, Cell Transformation, Viral genetics, DNA metabolism, Kirsten murine sarcoma virus genetics, Methylation, Molecular Sequence Data, Oligonucleotide Probes, Promoter Regions, Genetic, Rats, Transcription, Genetic, Genes, ras, Thyroglobulin genetics, Thyroid Gland metabolism, Trans-Activators antagonists & inhibitors
- Abstract
Exposure of rat thyroid cells for 1 week to a temperature-sensitive variant of Kirsten murine sarcoma virus (KiMSV) Ras inactivated the thyroglobulin promoter (pTg). Cellular dedifferentiation was paralleled by the loss of the thyroid-specific trans-acting factor, TgTF1, which binds to pTg. When Ras was denatured by shifting cells to 39 degrees C, TgTF1 binding and pTg function recovered rapidly without the synthesis of new protein. TgTF1 could be reactivated in vitro by treating nuclear extracts with protein kinase A. After 4 weeks of exposure to the oncogene, denaturation of Ras no longer restored TgTF1 binding or reactivated pTg. Incubation of nuclear extracts with protein kinase A likewise did not reactivate TgTF1. Cells chronically exposed to Ras did, however, yield differentiated clones after treatment with 5-azacytidine. We suggest that Ras induces dedifferentiation in two sequential steps: (1) Ras reduces PKA activity; TgTF1 (or an auxiliary protein) becomes dephosphorylated, and binding to pTg is abolished. (2) The effects of Ras become imprinted by methylation, possibly of the TgTF1 gene.
- Published
- 1991
- Full Text
- View/download PDF
33. The complete structure of the rat thyroglobulin gene.
- Author
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Musti AM, Avvedimento EV, Polistina C, Ursini VM, Obici S, Nitsch L, Cocozza S, and Di Lauro R
- Subjects
- Amino Acid Sequence, Animals, Base Sequence, Biological Evolution, Cattle, Chromosome Mapping, DNA genetics, Genes, Humans, Rats, Repetitive Sequences, Nucleic Acid, Thyroglobulin genetics
- Abstract
We have isolated the entire gene for rat thyroglobulin, the precursor for thyroid hormone biosynthesis. The gene is at least 170,000 base pairs (bp) long; 9000 bp of coding information are distributed in 42 exons of homogeneous size (150-200 bp) except for two exons of 1100 and 620 bp. The sequences coding for two major thyroxine-forming sites are localized in exons 2 and 39. These two sequences do not show any homology either at the DNA or at the protein-sequence level, even though they code for sites highly specialized for the same function. Furthermore, both the 3' and the 5' end of the thyroglobulin structural gene appear to be made of repetitive units, which again do not show any homology. On the basis of these observations, we propose that the thyroglobulin gene arose by shuffling of at least two segments, with different evolutionary histories, each of which already contained introns.
- Published
- 1986
- Full Text
- View/download PDF
34. Clusters of repeated sequences of chicken DNA are extensively methylated but contain specific undermethylated regions.
- Author
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Eden FC, Musti AM, and Sobieski DA
- Subjects
- 5-Methylcytosine, Animals, Cytosine analogs & derivatives, Cytosine analysis, DNA, Electrophoresis, Agar Gel, Genes, Methylation, Nucleic Acid Hybridization, Chickens genetics, Repetitive Sequences, Nucleic Acid
- Published
- 1981
- Full Text
- View/download PDF
35. Protein binding domains of the rat thyroglobulin promoter.
- Author
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Ursini MV, Gallo A, Olivetta E, and Musti AM
- Subjects
- Animals, Binding Sites, Rats, Thyroid Gland physiology, Transcription, Genetic, DNA-Binding Proteins metabolism, Promoter Regions, Genetic, Thyroglobulin genetics, Transcription Factors metabolism
- Abstract
We have previously shown that DNA elements controlling tissue specific expression of the rat thyroglobulin gene extend 170 bp upstream of the cap site and have identified a thyroid specific nuclear factor which binds the promoter in the -60 region (site C). Here we report that the distal portion of the promoter, extending from -160 to -120, contains two contiguous DNA elements (sites A and B) which interact with the same thyroid-specific factor binding to proximal site C. A second nuclear factor, ubiquitously distributed, binds to the distal site A. Transient cotransfection-competition studies show that all the three binding sites A, B and C titrate a trans-acting factor(s) which is necessary for the transcription of the thyroglobulin gene.
- Published
- 1989
- Full Text
- View/download PDF
36. A cell type specific factor recognizes the rat thyroglobulin promoter.
- Author
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Musti AM, Ursini VM, Avvedimento EV, Zimarino V, and Di Lauro R
- Subjects
- Animals, Base Composition, Base Sequence, Chromosome Deletion, Liver metabolism, Molecular Sequence Data, Mutation, Rats, Thyroid Gland metabolism, Transcription, Genetic, Cloning, Molecular, Genes, Promoter Regions, Genetic, Thyroglobulin genetics
- Abstract
We have fused a 900 base pair long DNA segment containing the transcriptional start site of the rat thyroglobulin (Tg) gene to the bacterial gene for chloramphenicol acetyltransferase (cat). The fusion gene has been introduced into three different cell lines derived from the rat thyroid gland and into a rat liver cell line. Expression of the fusion gene was detected only in the one thyroid cell line that is able to express the endogenous Tg gene. The minimum DNA sequence required for the cell type specific expression was determined by deletion analysis; it extends 170 nucleotides upstream of the transcription initiation site. The Tg promoter contains a readily detectable binding sites for a factor present in salt extracts of thyroid cell nuclei. This binding site is not recognized by the nuclear extracts of any other cell type that we have tested, suggesting that it may help mediate the cell type specific expression of the Tg gene.
- Published
- 1987
- Full Text
- View/download PDF
37. Repeated deoxyribonucleic acid clusters in the chicken genome contain homologous sequence elements in scrambled order.
- Author
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Musti AM, Sobieski DA, Chen BB, and Eden FC
- Subjects
- Animals, Bacteriophage lambda genetics, Base Sequence, Chickens, DNA, DNA Restriction Enzymes, Molecular Weight, Nucleic Acid Hybridization, Chromatin analysis, Cloning, Molecular, DNA, Recombinant metabolism
- Abstract
Part of the repeated deoxyribonucleic acid (DNA) in the chicken genome had a clustered organization. The following description of clustered repeated sequences is derived both from analysis of DNA segments cloned in lambda and from hybridization of individual cloned sequences to Southern blots of restricted total DNA. A cluster usually exceeds 20 kbp in length and consists principally, if not entirely, or repetitive DNA. Each cluster contains one cope of several different repeated sequences. The individual sequences occur several hundred times in the genome, but only once per cluster. Many of the clusters contain the same assortment of sequences but in scrambled order. In the genome, those repeated sequences that are elements of clusters occur mainly within the clustered context and are seldom, if ever, found as isolated elements flanked by nonrepeated DNA. These aspects of cluster organization suggest that the clustered sequences undergo limited rearrangement, maintaining the associations within clusters but allowing variability of sequence arrangement from cluster to cluster. The clusters that occupy the cloned DNA segments together represent at least 10% of the repetitive DNA of the chicken.
- Published
- 1981
- Full Text
- View/download PDF
38. Structural and physiological studies of the Escherichia coli histidine operon inserted into plasmid vectors.
- Author
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Bruni CB, Musti AM, Frunzio R, and Blasi F
- Subjects
- Base Sequence, Cloning, Molecular, DNA Restriction Enzymes, DNA, Bacterial analysis, DNA, Bacterial genetics, Genes, Genes, Regulator, Genetic Vectors, Escherichia coli genetics, Histidine biosynthesis, Operon, Plasmids
- Abstract
A fragment of deoxyribonucleic acid 5,300 base paris long and containing the promoter-proximal portion of the histidine operon of Escherichia coli K-12, has been cloned in plasmid pBR313 (plasmids pCB2 and pCB3). Restriction mapping, partial nucleotide sequencing, and studies on functional expression in vivo and on protein synthesis in minicells have shown that the fragment contains the regulatory region of the operon, the hisG, hisD genes, and part of the hisC gene. Another plasmid (pCB5) contained the hisG gene and part of the hisD gene. Expression of the hisG gene in the latter plasmid was under control of the tetracycline promoter of the pBR313 plasmid. The in vivo expression of the two groups of plasmids described above, as well as their effect on the expression of the histidine genes not carried by the plasmids but present on the host chromosome, has been studied. The presence of multiple copies of pCB2 or pCB3, but not of pCB5, prevented derepression of the chromosomal histidine operon. Possible interpretations of this phenomenon are discussed.
- Published
- 1980
- Full Text
- View/download PDF
39. Transcriptional mapping of two yeast genes coding for glyceraldehyde 3-phosphate dehydrogenase isolated by sequence homology with the chicken gene.
- Author
-
Musti AM, Zehner Z, Bostian KA, Paterson BM, and Kramer RA
- Subjects
- Animals, Base Sequence, Chickens genetics, Chromosome Mapping, DNA, Fungal biosynthesis, Glyceraldehyde-3-Phosphate Dehydrogenases biosynthesis, Nucleic Acid Hybridization, RNA, Fungal biosynthesis, Species Specificity, Transcription, Genetic, Glyceraldehyde-3-Phosphate Dehydrogenases genetics, Saccharomyces cerevisiae genetics
- Abstract
Homology between the coding regions of the chicken and yeast glyceraldehyde 3-phosphate dehydrogenase (GAPDH) genes was directly demonstrated by the hybridization of a cDNA clone coding for GAPDH in the chicken with EcoRI-digested yeast DNA. A yeast EcoRI fragment library in bacteriophage lambda was screened using the chicken cDNA plasmid as probe, and two recombinant phages were isolated, each one containing a different GAPDH gene. The initiation and termination sites for the GAPDH mRNA were localized for the two different GAPDH genes and compared to those of other yeast genes. Measurements of the relative mRNA levels for the two genes show that both genes are transcribed at about the same level when yeasts are grown on glucose media.
- Published
- 1983
- Full Text
- View/download PDF
40. Structural organization of the 3' half of the rat thyroglobulin gene.
- Author
-
Avvedimento VE, Musti AM, Obici S, Cocozza S, and Di Lauro R
- Subjects
- Animals, Base Composition, Base Sequence, DNA analysis, DNA Restriction Enzymes, Genetic Vectors, Nucleic Acid Hybridization, RNA, Messenger genetics, Rats, Cloning, Molecular, Genes, Thyroglobulin genetics
- Abstract
We report the structural organization of an 80 Kb segment of rat DNA, which encodes for about 40% of Thyroglobulin mRNA at the 3' end. The codogenic information included in this segment is splitted in 17 exons of homogeneous size (about 200 bp). The seven exons at the extreme 3' end have been precisely defined by DNA sequence analysis. No clear sequence homology is found among the exons, even though their coding capacity is quite similar, from 55 to 63 aminoacids residues. We located 2 hormonogenic (T4 forming) sites on the extreme 3' end of the gene in different exons. The DNA sequence coding for these functional sites shows a 70% homology in a 50 nucleotides segment. In addition we found a remnant of this sequence in other exons of the gene. Two large introns have been found on the 3' end of the gene: one is 17 Kb and the other one is more than 30 Kb long. On the basis of these findings and of preliminary studies on the remaining 5' end of the gene, we can predict that the minimum length of the rat TGB gene will be 150 Kb, which makes this gene the largest so far identified eukaryotic gene. We propose in addition that the 3' end exons arose by duplication of a common ancestor.
- Published
- 1984
- Full Text
- View/download PDF
41. The block of thyroglobulin synthesis, which occurs upon transformation of rat thyroid epithelial cells, is at the transcriptional level and it is associated with methylation of the 5' flanking region of the gene.
- Author
-
Berlingieri MT, Musti AM, Avvedimento VE, Di Lauro R, Di Fiore PP, and Fusco A
- Subjects
- Animals, Cell Line, DNA genetics, Epithelium metabolism, Kirsten murine sarcoma virus genetics, Methylation, Rats, Restriction Mapping, Thyroglobulin biosynthesis, Cell Transformation, Neoplastic, Genes, Regulatory Sequences, Nucleic Acid, Thyroglobulin genetics, Thyroid Gland metabolism, Transcription, Genetic
- Abstract
Transformation of rat thyroid epithelial cells by Kirsten murine sarcoma virus results in the block of certain thyroid differentiated functions, such as synthesis and secretion of thyroglobulin. Our studies, performed by a run-on assay, demonstrate that this block occurs at the transcriptional level. We also demonstrate the de novo methylation of two methylation-sensitive sites, located within the 5' end regulatory sequences of the thyroglobulin gene, in transformed cells, in the absence of any rearrangement of the gene. These two methylation-sensitive sites were methylated also in a rat thyroid cell line transformed by another retrovirus and in two normal cell lines which do not express the thyroglobulin gene.
- Published
- 1989
- Full Text
- View/download PDF
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