scholarly journals Activator protein-1 transactivation of the major immediate early locus is a determinant of cytomegalovirus reactivation from latency

2020 ◽  
Vol 117 (34) ◽  
pp. 20860-20867 ◽  
Author(s):  
Benjamin A. Krishna ◽  
Amanda B. Wass ◽  
Christine M. O’Connor
Keyword(s):  
Molecular Mechanisms ◽  
Viral Reactivation ◽  
Immediate Early ◽  
Activator Protein 1 ◽  
Activator Protein ◽  
Cytomegalovirus Reactivation ◽  
Immunological Stress ◽  
Distal Promoter ◽  
G Protein Coupled ◽  
Early Locus

Human cytomegalovirus (HCMV) is a ubiquitous pathogen that latently infects hematopoietic cells and has the ability to reactivate when triggered by immunological stress. This reactivation causes significant morbidity and mortality in immune-deficient patients, who are unable to control viral dissemination. While a competent immune system helps prevent clinically detectable viremia, a portrait of the factors that induce reactivation following the proper cues remains incomplete. Our understanding of the complex molecular mechanisms underlying latency and reactivation continues to evolve. We previously showed the HCMV-encoded G protein-coupled receptorUS28is expressed during latency and facilitates latent infection by attenuating the activator protein-1 (AP-1) transcription factor subunit, c-fos, expression and activity. We now show AP-1 is a critical component for HCMV reactivation. Pharmacological inhibition of c-fos significantly attenuates viral reactivation. In agreement, infection with a virus in which we disrupted the proximal AP-1 binding site in the major immediate early (MIE) enhancer results in inefficient reactivation compared to WT. Concomitantly, AP-1 recruitment to the MIE enhancer is significantly decreased following reactivation of the mutant virus. Furthermore, AP-1 is critical for derepression of MIE-driven transcripts and downstream early and late genes, while immediate early genes from other loci remain unaffected. Our data also reveal MIE transcripts driven from the MIE promoter, the distal promoter, and the internal promoter, iP2, are dependent upon AP-1 recruitment, while iP1-driven transcripts are AP-1–independent. Collectively, our data demonstrate AP-1 binding to and activation of the MIE enhancer is a key molecular process controlling reactivation from latency.

scholarly journals Activator protein-1 transactivation of the major immediate early locus is a determinant of cytomegalovirus reactivation from latency

2020 ◽  
Author(s):  
Benjamin A. Krishna ◽  
Amanda B. Wass ◽  
Christine M. O’Connor
Keyword(s):  
Transcription Factor ◽  
Human Cytomegalovirus ◽  
Molecular Mechanisms ◽  
Viral Reactivation ◽  
Immediate Early ◽  
Activator Protein 1 ◽  
Cellular Transcription Factor ◽  
Activator Protein ◽  
Cytomegalovirus Reactivation ◽  
Highly Active

AbstractHuman cytomegalovirus (HCMV) is a ubiquitous pathogen that latently infects hematopoietic cells and has the ability to reactivate when triggered by immunological stress. This reactivation causes significant morbidity and mortality in immune-deficient patients, who are unable to control viral dissemination. While a competent immune system helps prevent clinically detectable viremia, a portrait of the factors that induce reactivation following the proper cues remains incomplete. Our understanding of the complex molecular mechanisms underlying latency and reactivation continue to evolve. We previously showed the HCMV-encoded G-protein coupled receptor US28 is expressed during latency and facilitates latent infection by attenuating the activator protein-1 (AP-1) transcription factor subunit, c-fos, expression and activity. We now show AP-1 is a critical component for HCMV reactivation. Pharmacological inhibition of c-fos significantly attenuates viral reactivation. In agreement, infection with a virus in which we disrupted the proximal AP-1 binding site in the major immediate early (MIE) enhancer results in inefficient reactivation compared to wild type. Concomitantly, AP-1 recruitment to the MIE enhancer is significantly decreased following reactivation of the mutant virus. Further, AP-1 is critical for de-repression of MIE-driven transcripts and downstream early and late genes, while immediate early genes from other loci remain unaffected. Our data also reveal MIE transcripts driven from the MIE promoter, the distal promoter, and the internal promoter, iP2, are dependent upon AP-1 recruitment, while iP1-driven transcripts are AP-1-independent. Collectively, our data demonstrate AP-1 binding to and activation of the MIE enhancer is a key molecular process controlling reactivation from latency.Significance StatementHuman cytomegalovirus (HCMV) is a common pathogen that infects the majority of the population for life. This infection poses little threat in immunologically healthy individuals, but can be fatal in people with compromised immune systems. Our understanding of the mechanisms underlying latency and reactivation remains incomplete. Here, we show the cellular transcription factor, AP-1, is a key to regulating HCMV reactivation. Our findings reveal AP-1 binding to the major immediate early enhancer/promoter is critical for switching this locus from one that is repressed during latency to one that is highly active following reactivation. Our work provides a novel mechanism HCMV exploits to reactivate, highlighting AP-1 as a potential target to prevent HCMV reactivation.

scholarly journals The AP-1 Transcription Factor Is a Key Determinant of Human Cytomegalovirus Latency and Reactivation

Proceedings ◽  
2020 ◽  
Vol 50 (1) ◽  
pp. 127
Author(s):  
Benjamin A. Krishna ◽  
Amanda B. Wass ◽  
Christine M. O’Connor
Keyword(s):  
Transcription Factor ◽  
Human Cytomegalovirus ◽  
Infected Individual ◽  
Virus Production ◽  
Mapk Signaling ◽  
Viral Reactivation ◽  
Immediate Early ◽  
Activator Protein 1 ◽  
Latently Infected ◽  
Signaling Axis

Human cytomegalovirus (HCMV) is a ubiquitous pathogen that latently infects hematopoietic progenitor cells (HPCs). Individuals with a competent immune system are, for the most part, asymptomatic for the disease. However when a latently infected individual becomes immunosuppressed, HCMV can reactivate, causing severe morbidity and mortality. While much of the viral genome is transcriptionally silenced during latency, some genes are expressed, including the HCMV-encoded G-protein coupled receptor US28. We showed that US28 expression is required for latency, as it suppressed the activator protein-1 (AP-1) transcription factor by attenuating the AP-1 subunit, fos. In turn, this prevents AP-1 from binding and activating the major immediate early promoter (MIEP), the key promoter regulating the latent-to-lytic transcriptional “switch”. Our new data suggest that US28-mediated signaling during latency attenuates the Src-MAPK signaling axis to regulate AP-1. We find that US28 expression suppresses Src, MEK, and ERK, as well as fos phosphorylation and AP-1 binding to the MIEP. Conversely, the pharmacological inhibition of Src, MEK, or ERK in US28Δ-latently infected HPCs suppresses infectious virus production, demonstrating the important role for this signaling axis during latency. Our recent data also reveal that regulating AP-1 is a key determinant in balancing HCMV latency and reactivation. Infection with a virus in which we disrupted the proximal AP-1 binding site in the MIEP (AP-1Δp) leads to reduced AP-1 binding and inefficient viral reactivation compared to wild type. Furthermore, AP-1 is critical for the de-repression of MIEP-driven transcripts and downstream early and late genes, while other immediate early genes remain unaffected. Collectively, these data suggest that AP-1 binding to the MIEP is suppressed during latency, but is required for the efficient transactivation of the MIEP during reactivation. We are currently elucidating US28’s involvement in recruiting AP-1 to the MIEP during reactivation.

Protective role of silibinin on matrix metalloproteinase-2 and activator protein-1 on LoVo cells

2009 ◽  
Vol 27 (15_suppl) ◽  
pp. e15136-e15136
Author(s):  
C. Lin ◽  
Y. Chen
Keyword(s):  
Colon Cancer ◽  
Matrix Metalloproteinase ◽  
Molecular Mechanisms ◽  
Binding Activity ◽  
Matrix Metalloproteinase 2 ◽  
Lovo Cell ◽  
Activator Protein 1 ◽  
Activator Protein ◽  
Lovo Cells

e15136 Background: Silibinin, a flavonoid and the major active component of milk thistle, has been as a safe diet supplement for several decades. It has been proved with anti-hepatotoxic properties and pleiotropic anticancer capabilities. Current study aimed to investigate the role of silibinin as potential therapeutic target of colon cancer through antiangiogenesis and its related molecular mechanisms with matrix metalloproteinase- 2 (MMP-2) and activator protein-1 (AP-1). Colon cancer cell line, LoVo cells, treated with a major prognostic factor, interleukin-6 (IL-6), was studied. Methods and Results: By western blot analysis, silibinin suppressed MMP- 2 protein expression in time- and concentration-dependent manners. Furthermore, the inhibitors of JNK/AP-1 binding activity abolished the expression of MMP-2 in IL-6-stimulated LoVo cells, but not PI3K pathways. We also demonstrated that silibinin inhibited IL-6- stimulated LoVo cell migration and further tumor angiogenesis, which similar to the effects from addition with AP-1 inhibitor. By EMSA, the binding activity of AP-1 in LoVo cells was also decreased with silibinin treatment. In addition, the imaging of confocal microscopy revealed that AP-1 presentation was attenuated on IL-6-stimulated LoVo cells plus silibinin treatment. Conclusions: Taken together, these data indicated that silibinin inhibits angiogenesis through the suppression of MMP-2 expression and AP-1 binding activity in colon cancer cells. It suggests a novel anti-metastatic application of silibinin in colon cancer chemoprevention. No significant financial relationships to disclose.

scholarly journals Activator Protein-1 and Smad Proteins Synergistically Regulate Human Follicle-Stimulating Hormone β-Promoter Activity

Endocrinology ◽  
2008 ◽  
Vol 149 (11) ◽  
pp. 5577-5591 ◽  
Author(s):  
Ying Wang ◽  
Jérôme Fortin ◽  
Pankaj Lamba ◽  
Marco Bonomi ◽  
Luca Persani ◽  
...  
Keyword(s):  
Signaling Pathways ◽  
Promoter Activity ◽  
Molecular Mechanisms ◽  
Activin A ◽  
Dominant Negative ◽  
Activator Protein 1 ◽  
Reporter Activity ◽  
Activator Protein ◽  
Mapk Cascades ◽  
Smad Proteins

GnRH1 stimulates the synthesis and secretion of FSH and LH from the anterior pituitary gland. The molecular mechanisms through which GnRH1 produces these effects in humans have not been determined. Here, we examined transcriptional regulation of the human FSHβ (FSHB) subunit using reporter assays in immortalized murine gonadotrope cells. GnRH1 dose and time dependently stimulated FSHB promoter activity, with peak stimulation occurring at 8 h. GnRH1 rapidly stimulated various MAPK cascades, though the ERK1/2 and p38 pathways appeared to be most critical for FSHB induction. Indeed, constitutively active forms of both Raf1 kinase and MAP2K6 (MKK6) were sufficient to stimulate reporter activity. GnRH1 stimulated activator protein-1 (AP-1) (FosB, c-fos, JunB, and cJun) synthesis and complex formation, the latter of which bound to a conserved cis-element within −120 bp of the transcription start site. A second, lower affinity, site was mapped more proximally. Mutations of both cis-elements diminished GnRH1-stimulated promoter activity, though disruption of the higher affinity site had a more dramatic effect. A dominant-negative Fos protein dose dependently inhibited GnRH1-stimulated FSHB transcription, confirming a role for endogenous AP-1 proteins. MAPK kinase 1 (MEK1) and p38 inhibitors significantly attenuated GnRH1-stimulated c-fos, FosB, and JunB synthesis, suggesting a mechanism whereby the ERK1/2 and p38 signaling pathways regulate FSHB transcription. Activins and inhibins potently regulate FSH synthesis in rodents, but their roles in FSH regulation in humans are less clear. Activin A, though weak on its own, synergized with GnRH1 to stimulate human FSHB promoter activity. In contrast, activin A partially inhibited GnRH1-stimulated LHβ subunit (LHB) transcription. The GnRH1 and activin A signaling pathways appear to converge at the level of the high-affinity AP-1 site. Fos and Jun proteins synergistically regulate reporter activity through this element, and their effects are potentiated by coexpression of either Smad2 or Smad3, effectors in the activin signaling cascade. In summary, GnRH1 and activin A synergistically regulate human FSHB subunit transcription. The combined actions of AP-1 and Smad proteins acting through a conserved AP-1 element provide a candidate mechanism for this effect. The ability of activins to potentiate selectively the effects of GnRH1 on FSHB expression suggests a model for preferential increases in FSH secretion at the luteal-follicular transition of the menstrual cycle.

Phorbol ester treatment of human myometrial cells suppresses expression of oxytocin receptor through a mechanism that does not involve activator protein-1

2006 ◽  
Vol 291 (5) ◽  
pp. E922-E928 ◽  
Author(s):  
Allison Ball ◽  
Jing Wei Wang ◽  
Susan Wong ◽  
Barbara Zielnik ◽  
Jana Mitchell ◽  
...  
Keyword(s):  
Phorbol Ester ◽  
Prolonged Exposure ◽  
Late Pregnancy ◽  
Expression Vectors ◽  
Activator Protein 1 ◽  
Adenoviral Infection ◽  
Myometrial Cells ◽  
Activator Protein ◽  
Labor Onset ◽  
G Protein Coupled

Oxytocin (OT) is a potent uterine agonist. Its receptor (OTR) is a G protein-coupled receptor that is downregulated by prolonged exposure to OT. We hypothesized that activation of PKC mediated this OT-induced decrease in OTR expression. Diminished PKC activity in late pregnancy could underlie the increased expression of uterine OTR preceding labor onset. Using cell cultures of transformed human uterine myocytes, we determined the effects of PKC agonists and antagonists on the expression of OTR. We also explored the effects of overexpression of activator protein-1 (AP-1, a mediator of many PKC- and phorbol ester-induced effects) using adenoviral expression vectors for the AP-1 subunits c-Jun and c-Fos. Stimulation of PKC using the phorbol ester 12- O-tetradecanoylphorbol 13-acetate caused a rapid, significant (P ≤ 0.05) increase in c-Jun and c-Fos concentrations but a significant decrease in mRNA for OTR within 6 h followed by a significant decrease in OT binding by 24 h. Adenoviral infection of the cells with expression vectors for c-Jun and c-Fos increased the AP-1 subunits but had no effect on OTR expression. Furthermore, there were no changes in c-Fos or c-Jun levels in human intrauterine tissues around the time of labor onset, as measured by Western analyses. We conclude that phorbol ester treatment decreases OTR expression, likely through a mechanism that does not involve AP-1.

Cytokine synergy, collagenases and cartilage collagen breakdown

2003 ◽  
Vol 70 ◽  
pp. 125-133 ◽  
Author(s):  
Tim E. Cawston ◽  
Jenny M. Milner ◽  
Jon B. Catterall ◽  
Andrew D. Rowan
Keyword(s):  
Matrix Metalloproteinases ◽  
Inflammatory Cytokines ◽  
Activator Protein 1 ◽  
Activator Protein ◽  
And Cartilage ◽  
Pro Inflammatory Cytokines

We have investigated proteinases that degrade cartilage collagen. We show that pro-inflammatory cytokines act synergistically with oncastatin M to promote cartilage collagen resorption by the up-regulation and activation of matrix metalloproteinases (MMPs). The precise mechanisms are not known, but involve the up-regulation of c-fos, which binds to MMP promoters at a proximal activator protein-1 (AP-1) site. This markedly up-regulates transcription and leads to higher levels of active MMP proteins.

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