Matrix metalloproteinase-3 induction in rat brain astrocytes: focus on the role of two AP-1 elements

2008 ◽  
Vol 410 (3) ◽  
pp. 605-611 ◽  
Author(s):  
Kwang Soo Kim ◽  
Hee Young Kim ◽  
Eun-hye Joe ◽  
Ilo Jou
Keyword(s):  
Rat Brain ◽  
Interferon Γ ◽  
Site Directed Mutagenesis ◽  
Start Codon ◽  
Activator Protein 1 ◽  
Mobility Shift ◽  
Matrix Metalloproteinase 3 ◽  
Mitogen Activated Protein ◽  
Electrophoretic Mobility Shift Assays

Many brain cells secrete MMPs (matrix metalloproteinases), and increased or misregulated MMP levels are found in neurodegenerative disorders. Here we report that MMP-3 transcription and protein secretion were increased in rat brain astrocytes stimulated with lipopolysaccharide, gangliosides or interferon-γ. Sequential deletion of the MMP-3 promoter revealed that sequences between −0.5 kb and the start codon were crucial for the transcriptional induction of MMP-3. In addition, experiments using pharmacological inhibitors of individual mitogen-activated protein kinases revealed that MMP-3 induction and promoter activity involved Jun N-terminal kinase, a representative upstream signal of AP-1 (activator protein-1). Sequence analyses of the region of the MMP-3 promoter 500 bp from the start codon indicated the presence of three AP-1 binding sequences. Among them, electrophoretic-mobility-shift assays as well as site-directed mutagenesis of individual AP-1 sequences revealed that distal and middle, but not proximal, sequences largely mediated its induction. Together, these results indicate that AP-1 could control MMP-3 induction in brain astrocytes and that its regulation through specific AP-1 elements could be exploited in the treatment of brain pathologies in which increased expression of MMP-3 plays crucial roles.

Distinct functional contributions of 2 GABP–NRF-2 recognition sites within the context of the human TOMM70 promoter

2006 ◽  
Vol 84 (5) ◽  
pp. 813-822 ◽  
Author(s):  
José R. Blesa ◽  
José Hernández-Yago
Keyword(s):  
Chromatin Immunoprecipitation ◽  
Binding Sites ◽  
Site Directed Mutagenesis ◽  
Outer Mitochondrial Membrane ◽  
Mobility Shift ◽  
A Subunit ◽  
Expression Evidence ◽  
Electrophoretic Mobility Shift Assays

TOMM70 is a subunit of the outer mitochondrial membrane translocase that plays a major role as a receptor of hydrophobic preproteins targeted to mitochondria. We have previously reported 2 binding sites for the transcription factor GABP–NRF-2 in the promoter region of the human TOMM70 gene that are important in activating transcription. To assess the functionality and actual role of these sites, chromatin immunoprecipitation, site-directed mutagenesis, and electrophoretic mobility shift assays were carried out. We conclude that GABP–NRF-2 binds in vivo to the TOMM70 promoter, and that the 2 GABP–NRF-2 binding sites of the promoter have different functional contributions in promoting TOMM70 expression. Evidence is provided that they work in an additive manner as single sites.

scholarly journals Silencing of the Gene for the α-Subunit of Human Chorionic Gonadotropin by the Embryonic Transcription Factor Oct-3/4

1997 ◽  
Vol 11 (11) ◽  
pp. 1651-1658 ◽  
Author(s):  
Limin Liu ◽  
Douglas Leaman ◽  
Michel Villalta ◽  
R. Michael Roberts
Keyword(s):  
Transcription Factor ◽  
Binding Site ◽  
Gene Promoter ◽  
Site Directed Mutagenesis ◽  
Mobility Shift ◽  
Α Subunit ◽  
Choriocarcinoma Cells ◽  
Human Choriocarcinoma Cells ◽  
Electrophoretic Mobility Shift Assays ◽  
Jar Cells

Abstract CG is required for maintenance of the corpus luteum during pregnancy in higher primates. As CG is a heterodimeric molecule, some form of coordinated control must be maintained over the transcription of its two subunit genes. We recently found that expression of human CG β-subunit (hCGβ) in JAr human choriocarcinoma cells was almost completely silenced by the embryonic transcription factor Oct-3/4, which bound to a unique ACAATAATCA octameric sequence in the hCGβ gene promoter. Here we report that Oct-3/4 is also a potent inhibitor of hCG α-subunit (hCGα) expression in JAr cells. Oct-3/4 reduced human GH reporter expression from the −170 hCGα promoter in either the presence or absence of cAMP by about 70% in transient cotransfection assays, but had no effect on expression from either the −148 hCGα or the −99 hCGα promoter. Unexpectedly, no Oct-3/4-binding site was identified within the −170 to −148 region of the hCGα promoter, although one was found around position −115 by both methylation interference footprinting and electrophoretic mobility shift assays. Site-directed mutagenesis of this binding site destroyed the affinity of the promoter for Oct-3/4, but did not affect repression of the promoter. Therefore, inhibition of hCGα gene transcription by Oct-3/4 appears not to involve direct binding of this factor to the site responsible for silencing. When stably transfected into JAr cells, Oct-3/4 reduced the amounts of both endogenous hCGα mRNA and protein by 70–80%. Oct-3/4 is therefore capable of silencing both hCGα and hCGβ gene expression. We suggest that as the trophoblast begins to form, reduction of Oct-3/4 expression permits the coordinated onset of transcription from the hCGα and hCGβ genes.

scholarly journals Phloretin alleviates dinitrochlorobenzene-induced dermatitis in BALB/c mice

2020 ◽  
Vol 34 ◽  
pp. 205873842092944
Author(s):  
Chieh-Shan Wu ◽  
Shih-Chao Lin ◽  
Shiming Li ◽  
Yu-Chih Chiang ◽  
Nicole Bracci ◽  
...  
Keyword(s):  
Mouse Model ◽  
Serum Levels ◽  
Interferon Γ ◽  
Chronic Inflammatory Disease ◽  
Mitogen Activated Protein Kinase ◽  
Normal Ranges ◽  
Mitogen Activated Protein ◽  
Ifn Γ ◽  
Allergic Contact

Atopic dermatitis (AD) is a chronic inflammatory disease of the skin that substantially affects a patient’s quality of life. While steroids are the most common therapy used to temporally alleviate the symptoms of AD, effective and nontoxic alternatives are urgently needed. In this study, we utilized a natural, plant-derived phenolic compound, phloretin, to treat allergic contact dermatitis (ACD) on the dorsal skin of mice. In addition, the effectiveness of phloretin was evaluated using a mouse model of ACD triggered by 2,4-dinitrochlorobenzene (DNCB). In our experimental setting, phloretin was orally administered to BALB/c mice for 21 consecutive days, and then, the lesions were examined histologically. Our data revealed that phloretin reduced the process of epidermal thickening and decreased the infiltration of mast cells into the lesion regions, subsequently reducing the levels of histamine and the pro-inflammatory cytokines interleukin (IL)-6, IL-4, thymic stromal lymphopoietin (TSLP), interferon-γ (IFN-γ) and IL-17A in the serum. These changes were associated with lower serum levels after phloretin treatment. In addition, we observed that the mitogen-activated protein kinase (MAPK) and NF-κB pathways in the dermal tissues of the phloretin-treated rodents were suppressed compared to those in the AD-like skin regions. Furthermore, phloretin appeared to limit the overproliferation of splenocytes in response to DNCB stimulation, reducing the number of IFN-γ-, IL-4-, and IL-17A-producing CD4+ T cells in the spleen back to their normal ranges. Taken together, we discovered a new therapeutic role of phloretin using a mouse model of DNCB-induced ACD, as shown by the alleviated AD-like symptoms and the reversed immunopathological effects. Therefore, we believe that phloretin has the potential to be utilized as an alternative therapeutic agent for treating AD.

scholarly journals Modulation of covR Expression in Streptococcus mutans UA159

2008 ◽  
Vol 190 (13) ◽  
pp. 4478-4488 ◽  
Author(s):  
Patrick Chong ◽  
Laura Drake ◽  
Indranil Biswas
Keyword(s):  
Dental Caries ◽  
Streptococcus Mutans ◽  
Growth Phase ◽  
Response Regulator ◽  
Transcriptional Start Site ◽  
Site Directed Mutagenesis ◽  
Start Codon ◽  
Pcr Analysis ◽  
Exponential Growth Phase ◽  
Mobility Shift

ABSTRACT The biofilm-forming Streptococcus mutans is a gram-positive bacterium that resides in the human oral cavity and is considered to be the primary etiological agent in the formation of dental caries. The global response regulator CovR, which lacks a cognate sensor kinase, is essential for the pathogenesis and biofilm formation of this bacterium, but it is not clear how covR expression is regulated in S. mutans. In this communication, we present the results of our studies examining various factors that regulate the expression of covR in S. mutans UA159. The results of Southern hybridization and PCR analysis indicated that CovR is an orphan response regulator in various isolates of S. mutans. The transcriptional start site for covR was found to be 221 base pairs upstream of the ATG start codon, and site-directed mutagenesis of the upstream TATAAT box confirmed our findings. The expression of covR is growth phase dependent, with maximal expression observed during exponential-growth phase. While changes to the growth temperature did not significantly affect the expression of covR, increasing the pH or the concentration of Mg2+ in the growth medium leads to an increase in covR expression. The results of semiquantitative reverse transcriptase PCR analysis and in vivo transcriptional-fusion reporter assays indicated that CovR autoregulates its own expression; this was verified by the results of electrophoretic mobility shift assays and DNase I protection assays, which demonstrated direct binding of CovR to the promoter region. Apparently, regulation by Mg2+ and the autoregulation of covR are not linked. A detailed analysis of the regulation of CovR may lead to a better understanding of the pathogenesis of S. mutans, as well as providing further insight into the prevention of dental caries.

Role of Cjun in Gγ-Globin Gene Trans-Activation.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1870-1870
Author(s):  
Sirisha Kodeboyina ◽  
Sima Zein ◽  
Moosueng Lee ◽  
Parimaladevi Balamurugan ◽  
Xiao Yao ◽  
...  
Keyword(s):  
Electrophoretic Mobility ◽  
Globin Gene ◽  
Fetal Hemoglobin ◽  
K562 Cells ◽  
Luciferase Reporter ◽  
Complete Analysis ◽  
Dependent Manner ◽  
Mobility Shift ◽  
Electrophoretic Mobility Shift Assays

Abstract Previous studies from our laboratory demonstrated the role of the G-CRE (Gγ-globin cAMP response element) in drug-mediated fetal hemoglobin induction. The G-CRE located at −1222 to −1229 in the promoter of Gγ-globin gene, contains binding site for trans-factors CREB1, ATF-2 and cJun. We previously demonstrated binding of phosphorylated CREB1 and ATF-2 to this element via p38 MAPK signaling triggered by sodium butyrate (NaB) and trichostatin A (TSA). Electrophoretic mobility shift assays with a probe containing the AC → TG mutation in the G-CRE (TGTGGTCA, m2) abolished trans-factor binding to the G-CRE. Furthermore, Gγ promoter activity was abolished in the PGL3 luciferase reporter vector driven by the Gγ promoter (−1500 to +36) carrying the m2 mutation. (Sangerman et al. Blood108:3590–9, 2006). Subsequent studies in our laboratory were aimed at understanding the role of trans-factor cJun, an AP-1 family member, as a regulator of Gγ-globin expression via the G-CRE site. In K562 cells treated with 2mM NaB or 0.3μM TSA for 48 hrs, cJun phosphorylation increased 2.8-fold and 6.4-fold respectively by western blot analysis. Chromatin immunoprecipitation studies showed 16-fold chromatin enrichment in the −1225 Gγ-globin region compared to IgG control studies indicative of significant cJun binding in vivo at steady state. Electrophoretic mobility shift assays using cJun monoclonal antibody demonstrated a supershifted DNA-protein complex confirming binding of cJun to the G-CRE probe. To gain evidence for a functional role of cJun, we performed enforced expression studies using the pLen-cJun vector. In a concentration dependent manner, over-expression of cJun increased luciferase activity up to 350-fold in the luciferase reporter plasmid controlled by the Gγ-promoter (−1500 to +36). As predicted from binding studies, the m2 mutation in this promoter abolished the cJunmediated trans-activation confirming that the G-CRE is required to mediate effects of cJun. We are currently investigating the ability of cJun to trans-activate the endogenous Gγ-globin gene in K562 cells. To achieve this goal, K562 stable lines were established with the expression vectors pLen-cJun and empty vector. A complete analysis of the stable lines is in progress. Future investigations to identify other components of the functional CREB1/ATF2/cJun enhanceosome complex bound to the G-CRE will be performed using affinity chromatography and mass spectrometry. This information will be used to develop strategies for fetal hemoglobin induction.

Detection of a Complex That Associates With the Bβ Fibrinogen G−455-A Polymorphism

Blood ◽  
1998 ◽  
Vol 92 (9) ◽  
pp. 3286-3293
Author(s):  
Erika T. Brown ◽  
Gerald M. Fuller
Keyword(s):  
Luciferase Reporter ◽  
Molecular Evidence ◽  
Luciferase Reporter Gene ◽  
Nucleotide Substitutions ◽  
Mobility Shift ◽  
Binding Characteristics ◽  
Base Sequences ◽  
American Society ◽  
Electrophoretic Mobility Shift Assays

The promoter region of the Bβ fibrinogen gene containing the polymorphic site (G−455-A) shows an increase in fibrinogen levels for individuals containing an adenine rather than a guanine. Two methods were used to explore the possible functional role of this region. Electrophoretic mobility shift assays (EMSAs) were performed using specific DNA probes containing base sequences pertinent to the allelic site. Specific DNA binding proteins were detected and their binding characteristics were determined. Secondly, we placed DNA fragments containing different −455 nucleotide substitutions of the Bβ promoter upstream of a luciferase reporter gene and transfected them into HepG2 cells to determine their effect on transactivation. An adenine at position −455 resulted in greater luciferase activity than when a guanine was present. UV cross-linking bound protein to the DNA demonstrated a 47-kD protein binding preferentially to the site when a guanine rather than an adenine was present at −455. We hypothesize that a transactivation protein complex associates with the site, but its association is stronger when guanine is present, thereby slowing downstream Bβ gene transcription. These data provide the first molecular evidence that accounts for the increase in fibrinogen in individuals carrying this allele.© 1998 by The American Society of Hematology.

Transcriptional coactivation of c-Jun by the KSHV-encoded LANA

Blood ◽  
2004 ◽  
Vol 103 (1) ◽  
pp. 222-228 ◽  
Author(s):  
Jiabin An ◽  
Yiping Sun ◽  
Matthew B. Rettig
Keyword(s):  
Primary Effusion Lymphoma ◽  
Kaposi Sarcoma ◽  
Nuclear Antigen ◽  
Physical Interaction ◽  
Activator Protein 1 ◽  
Transcriptional Coactivator ◽  
Mobility Shift ◽  
Associated Diseases ◽  
293 Cells ◽  
Electrophoretic Mobility Shift Assays

Abstract The Kaposi sarcoma–associated herpesvirus (KSHV)–encoded latency-associated nuclear antigen (LANA) modulates viral and cellular gene expression, including interleukin 6 (IL-6), a growth factor for KSHV-associated diseases. LANA-driven IL-6 expression is dependent on the activator protein 1 (AP1) response element (RE) within the IL-6 promoter. We show that LANA activates the AP1 RE in a Jun-dependent fashion and that LANA enhances the transcriptional activity of a GAL4-Jun fusion protein. Coimmunoprecipitation studies documented a physical interaction between LANA and c-Jun in transiently transfected 293 cells as well as the KSHV-infected BCBL-1 primary effusion lymphoma (PEL) cell line. Taken together, these data indicate that LANA is a transcriptional coactivator of c-Jun. In addition, electrophoretic mobility shift assays demonstrated that LANA induces binding of a c-Jun-Fos heterodimer to the AP1 RE, but does not itself bind to the AP1 RE. RNA interference experiments confirmed that LANA activates the AP1 RE, stimulates binding of a c-Jun-Fos heterodimer to the AP1 RE, and induces expression of IL-6. These data indicate that LANA is a transcriptional coactivator of c-Jun, a function that may have implications for the pathogenesis of KSHV-associated diseases.

scholarly journals The Sin3p PAH Domains Provide Separate Functions Repressing Meiotic Gene Transcription in Saccharomyces cerevisiae

2010 ◽  
Vol 9 (12) ◽  
pp. 1835-1844 ◽  
Author(s):  
Michael J. Mallory ◽  
Michael J. Law ◽  
Lela E. Buckingham ◽  
Randy Strich
Keyword(s):  
Cell Division ◽  
Vegetative Growth ◽  
Mitotic Cell ◽  
Amphipathic Helix ◽  
Mobility Shift ◽  
Mitotic Cell Division ◽  
Meiotic Gene ◽  
Electrophoretic Mobility Shift Assays ◽  
Promoter Interaction

ABSTRACT Meiotic genes in budding yeast are repressed during vegetative growth but are transiently induced during specific stages of meiosis. Sin3p represses the early meiotic gene (EMG) by bridging the DNA binding protein Ume6p to the histone deacetylase Rpd3p. Sin3p contains four paired amphipathic helix (PAH) domains, one of which (PAH3) is required for repressing several genes expressed during mitotic cell division. This report examines the roles of the PAH domains in mediating EMG repression during mitotic cell division and following meiotic induction. PAH2 and PAH3 are required for mitotic EMG repression, while electrophoretic mobility shift assays indicate that only PAH2 is required for stable Ume6p-promoter interaction. Unlike mitotic repression, reestablishing EMG repression following transient meiotic induction requires PAH3 and PAH4. In addition, the role of Sin3p in reestablishing repression is expanded to include additional loci that it does not control during vegetative growth. These findings indicate that mitotic and postinduction EMG repressions are mediated by two separate systems that utilize different Sin3p domains.

MAPK-induced Ser727 phosphorylation promotes SUMOylation of STAT1

2007 ◽  
Vol 409 (1) ◽  
pp. 179-185 ◽  
Author(s):  
Sari Vanhatupa ◽  
Daniela Ungureanu ◽  
Maija Paakkunainen ◽  
Olli Silvennoinen
Keyword(s):  
Interferon Γ ◽  
Dominant Negative ◽  
Mitogen Activated Protein Kinase ◽  
Extracellular Signal Regulated Kinase ◽  
Extracellular Signal ◽  
Microbial Infections ◽  
Mitogen Activated Protein ◽  
P38mapk Pathway ◽  
Ifn Γ

STAT1 (signal transducer and activator of transcription 1) is a critical mediator of IFN-γ (interferon-γ)-induced gene responses, and its function is regulated through phosphorylation of Tyr701 and Ser727. MAPK (mitogen-activated protein kinase) pathways mediate phosphorylation of Ser727 in response to microbial infections, stress stimuli and growth factors. Recently, STAT1 was found to become modified by PIAS (protein inhibitor of activated STAT)-mediated SUMO-1 (small ubiquitin-related modifier-1) conjugation at Lys703, but the regulation of this modification is largely unknown. Here, we have investigated the role of MAPK-induced Ser727 phosphorylation in regulation of STAT1 SUMOylation. Activation of the p38MAPK pathway by upstream activating kinase, MKK6 (MAPK kinase-6) or osmotic stress enhanced the SUMOylation of STAT1, which was counteracted by the p38MAPK inhibitor SB202190 or by dominant-negative p38MAPK. Activation of the ERK1/2 (extracellular-signal-regulated kinase 1/2) pathway by Raf-1 also enhanced Ser727 phosphorylation and SUMOylation of STAT1, and this induction was counteracted by PD98059 inhibitor. Mutation of Ser727 to alanine abolished the p38MAPK-induced SUMOylation. Furthermore, S727D and S727E mutations, which mimic the phosphorylation of Ser727, enhanced the basal SUMOylation of STAT1 and interaction between PIAS1 and STAT1. Taken together, these results identify Ser727 phosphorylation as a regulator of STAT1 SUMOylation and highlight the central role of Ser727 in co-ordination of STAT1 functions in cellular responses.

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