An official website of the United States government
Conserved NF-κB signaling pathways shape immune responses in animals. In mammals, NF-κB activation patterns and downstream transcription vary with stimulus, cell type, and stochastic differences among identically treated cells. Whether animals without adaptive immunity exhibit similar heterogeneity or rely on distinct immune strategies remains unknown. We engineered Drosophila melanogaster S2* reporter cells as an immune-responsive model to monitor the dynamics of an NF-κB transcription factor, Relish, and downstream transcription in single, living cells. Following immune stimulation, Relish exhibits diverse nuclear localization dynamics that fall into distinct categories, with both the fraction of responsive cells and their activation speed rising with stimulus dose. Pre-stimulus features, including Relish nuclear fraction, predict a cell's responsiveness to stimulation. Simultaneous measurement of Relish and downstream transcription revealed that the probability of transcriptional bursts from immune-responsive enhancers correlates with Relish nuclear fraction. The number of NF-κB binding sites tunes transcriptional activity among immune enhancers. Our study uncovers heterogeneity in NF-κB activation and target gene expression within Drosophila, illustrating how dynamic NF-κB behavior and enhancer architecture tune gene regulation.
more »
« less
Coumarinolignans with Reactive Oxygen Species (ROS) and NF-κB Inhibitory Activities from the Roots of Waltheria indica
Seven new coumarinolignans, walthindicins A–F (1a, 1b, 2–5, 7), along with five known analogs (6, 8–11), were isolated from the roots of Waltheria indica. The structures of the new compounds are determined by detailed nuclear magnetic resonance (NMR), circular dichroism (CD) with extensive computational support, and mass spectroscopic data interpretation. Compounds were tested for their antioxidant activity in Human Cervical Cancer cells (HeLa cells). Compounds 1a and 6 showed higher reactive oxygen species (ROS) inhibitory activity at 20 μg/mL when compared with other natural compound-based antioxidants such as ascorbic acid. Considering the role of ROS in nuclear-factor kappa B (NF-κB) activation, compounds 1a and 6 were evaluated for NF-κB inhibitory activity and showed a concentration-dependent inhibition in Human Embryonic Kidney 293 cells (Luc-HEK-293).
more »
« less
- Award ID(s):
- 1920304
- PAR ID:
- 10373846
- Date Published:
- Journal Name:
- Molecules
- Volume:
- 27
- Issue:
- 10
- ISSN:
- 1420-3049
- Page Range / eLocation ID:
- 3270
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
-
null (Ed.)The transcriptional co-activator YAP1 (yes-associated protein 1) is a critical nuclear effector of the Hippo pathway. The Hippo pathway regulates cell growth, cell motility, cell migration, and carcinogenesis, but poorly defined mechanism. We investigated biochemical and functional interactions between YAP1 and the nuclear factor (NF)-kappa B/RELA subunit in prostate cancer cell models. We demonstrated that endogenous YAP1 and RELA form protein complexes in the cell, as revealed by co-immunoprecipitation and western blotting. Compared with control, we found that combined treatment of cells with androgen and SDF-1a (stromal cell-derived factor-1 alpha) or RANKL (receptor activator of NF-kappa B ligand) enhanced the protein-protein interaction between YAP1 and RELA, as showed by proximity ligation assay. Our confocal microscopy experiment further showed that combined SDF-1aand androgen treatment promoted the YAP1 and RELA colocalization instead of single-agent treatment. Moreover, our promoter-reporter and RNAi experiments showed that knockdown of YAP1 or TEAD, a key mediator of the YAP transcription, significantly reduced the NF-Kappa B promoter-reporter gene activity. Also, disruption of YAP1 activity attenuated the TEAD-RELA interaction. Furthermore, the controlled expression of MST1/STK4, a potent inhibitor of YAP1, attenuated the NF-Kappa B-promoter reporter activity. Additionally, our unbiased bioinformatics analysis of the exiting ChIP-seq (chromatin immunoprecipitation-sequencing) data sets identified several genes that are likely co-regulated by the YAP1/TEAD and NF-Kappa B/RELA transcription factors. These findings suggest that cooperative androgen and cytokine signaling regulates Hippo/YAP and NF-Kappa B interaction. Thus, the YAP1/TEAD and NF-Kappa B/RELA interaction may have critical roles in cellular biology and human diseases.more » « less
-
Abstract We provide a functional characterization of transcription factor NF-κB in protists and provide information about the evolution and diversification of this biologically important protein. We characterized NF-κB in two protists using phylogenetic, cellular, and biochemical techniques. NF-κB of the holozoanCapsaspora owczarzaki(Co) has an N-terminal DNA-binding domain and a C-terminal Ankyrin repeat (ANK) domain, and its DNA-binding specificity is more similar to metazoan NF-κB proteins than to Rel proteins. Removal of the ANK domain allowsCo-NF-κB to enter the nucleus, bind DNA, and activate transcription. However, C-terminal processing ofCo-NF-κB is not induced by IκB kinases in human cells. OverexpressedCo-NF-κB localizes to the cytoplasm inCocells.Co-NF-κB mRNA and DNA-binding levels differ across threeCapsasporalife stages. RNA-sequencing and GO analyses identify possible gene targets ofCo-NF-κB. Three NF-κB-like proteins from the choanoflagellateAcanthoeca spectabilis(As) contain conserved Rel Homology domain sequences, but lack C-terminal ANK repeats. All threeAs-NF-κB proteins constitutively enter the nucleus of cells, but differ in their DNA-binding abilities, transcriptional activation activities, and dimerization properties. These results provide a basis for understanding the evolutionary origins of this key transcription factor and could have implications for the origins of regulated immunity in higher taxa.more » « less
-
Microglia transform in response to changes in sensory or neural activity, such as sensory deprivation. However, little is known about how specific frequencies of neural activity, or brain rhythms, affect microglia and cytokine signaling. Using visual noninvasive flickering sensory stimulation (flicker) to induce electrical neural activity at 40 hertz, within the gamma band, and 20 hertz, within the beta band, we found that these brain rhythms differentially affect microglial morphology and cytokine expression in healthy animals. Flicker induced expression of certain cytokines independently of microglia, including interleukin-10 and macrophage colony-stimulating factor. We hypothesized that nuclear factor κB (NF-κB) plays a causal role in frequency-specific cytokine and microglial responses because this pathway is activated by synaptic activity and regulates cytokines. After flicker, phospho–NF-κB colabeled with neurons more than microglia. Inhibition of NF-κB signaling down-regulated flicker-induced cytokine expression and attenuated flicker-induced changes in microglial morphology. These results reveal a mechanism through which brain rhythms affect brain function by altering microglial morphology and cytokines via NF-κB.more » « less
-
HIV latency regulation in monocytes and macrophages can vary according to signals directing differentiation, polarization, and function. To investigate these processes, we generated an HIV latency model in THP-1 monocytes and showed differential levels of HIV reactivation among clonal populations. Monocyte-to-macrophage differentiation of HIV-infected primary human CD14+ and THP-1 cells induced HIV reactivation and showed that virus production increased concomitant with macrophage differentiation. We applied the HIV-infected THP-1 monocyte-to-macrophage (MLat) model to assess the biological mechanisms regulating HIV latency dynamics during monocyte-to-macrophage differentiation. We pinpointed protein kinase C signaling pathway activation and Cyclin T1 upregulation as inherent differentiation mechanisms that regulate HIV latency reactivation. Macrophage polarization regulated latency, revealing proinflammatory M1 macrophages suppressed HIV reactivation while anti-inflammatory M2 macrophages promoted HIV reactivation. Because macrophages rely on reactive-oxygen species (ROS) to exert numerous cellular functions, we disrupted redox pathways and found that inhibitors of the thioredoxin (Trx) system acted as latency-promoting agents in T-cells and monocytes, but opposingly acted as latency-reversing agents in macrophages. We explored this mechanism with Auranofin, a clinical candidate for reducing HIV reservoirs, and demonstrated Trx reductase inhibition led to ROS induced NF-κB activity, which promoted HIV reactivation in macrophages, but not in T-cells and monocytes. Collectively, cell type-specific differences in HIV latency regulation could pose a barrier to HIV eradication strategies.more » « less
- Free Publicly Accessible Full Text
-
Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.3390/molecules27103270
