Honey gold nanoparticles attenuate the secretion of IL-6 by LPS-activated macrophages.

Interleukin-6 (IL-6) is a pleiotropic cytokine that coordinates host immune responses to infection. Though essential to the acute phase response, prolonged IL-6-mediated recruitment of mononuclear cells has been implicated in chronic inflammatory diseases such as rheumatoid arthritis, psoriasis, and Crohn's disease. Accordingly, identifying novel therapeutics that diminish circulating IL-6 levels could benefit individuals suffering from chronic inflammation. In immunocompetent hosts, bacterial lipopolysaccharide (LPS) recognition by toll-like receptor 4 (TLR4) activates the transcription factor NF-κB, driving macrophage production of IL-6. Interestingly, both citrate-stabilized and 'green' synthesized gold nanoparticles (AuNPs) have been shown to modulate the cytokine responses of LPS-activated macrophages. Here we demonstrate that AuNPs, synthesized with commercial and locally sourced honey, downregulate LPS-induced macrophage secretion of IL-6. Compared to LPS-only controls, inhibition of IL-6 levels was observed for all three types of honey AuNPs. The effect was likely driven by honey AuNP-mediated perturbation of the TLR4/NF-κB signaling pathway, as evidenced by a reduction in the phosphorylation of IκB. Further investigation into the anti-inflammatory properties of honey AuNPs may yield novel therapeutics for the treatment of chronic inflammation.

Isoform specific knockdown of the ETS transcription factor Pointed in Drosophila S2 cells.

Alternate splicing of the pointed ( pnt ) gene locus produces two major isoforms, PntP1 and PntP2. Understanding their individual contributions to key developmental processes and identification of their genome-wide transcriptional targets has been hampered by a number of factors including their essential roles during embryonic development, and co-expression in several tissues. siRNAs were designed to target isoform-specific exons that code for the unique N-terminal region of either PntP1 or PntP2. The efficacy and specificity of the siRNAs were examined by co-transfection of isoform specific siRNAs with plasmids encoding epitope tagged PntP1 or PntP2 in Drosophila S2 cells. All P1-specific siRNAs were demonstrated to knockdown PntP1 protein level to greater than 95%, while having nominal impact on PntP2 level. Similarly, PntP2 siRNAs while ineffective at eliminating PntP1, were shown to reduce PntP2 protein level by 87-99%.

The effectiveness of EGFR knockdown by RNAi lines varies depending on the tissue.

In Drosophila , the Epidermal growth factor receptor (EGFR) signaling pathway is known to be critically involved in multiple stages of development. We induced a loss of function phenotype in the eyes, wings, and somatic follicle cells using four EGFR RNAi lines: HMS05003 and JF02283, which produce short hairpin RNAs, as well as JF01368 and KK100051, which produce long hairpin RNAs. Using these four lines, we completed a systematic comparison of the ability of short hairpin vs long hairpin RNAi lines to produce loss-of-function phenotypes in the above-mentioned tissues. Tissue specific knockdown was achieved by using Gal4 drivers specific to the three tissues being studied. In the eyes and wings, the KK100051 line induced the most severe phenotype, while the JF01368 line was the least severe, but in the somatic follicle cells, the KK100051 line was the least effective, while the JF01368 and JF02283 lines were comparable with respect to phenotypic severity. We conclude that there is significant tissue specific variability exhibited by the different RNAi lines.

Lessons from Drosophila Pointed, an ETS family transcription factor and key nuclear effector of the RTK signaling pathway.

The ETS family of transcription factors are evolutionarily conserved throughout the metazoan lineage and are critical for regulating cellular processes such as proliferation, differentiation, apoptosis, angiogenesis, and migration. All members have an ETS DNA binding domain, while a subset also has a protein-protein interaction domain called the SAM domain. Pointed (Pnt), an ETS transcriptional activator functions downstream of the receptor tyrosine kinase (RTK) signaling pathway to regulate diverse processes during the development of Drosophila. This review highlights the indispensable role that Pnt plays in regulating normal development and how continued investigation into its function and regulation will provide key mechanistic insight into understanding why the de-regulation of its vertebrate orthologs, ETS1 and ETS2 results in cancer.

Short hairpin RNA is more effective than long hairpin RNA in eliciting pointed loss-of-function phenotypes in Drosophila.

Pointed (Pnt) is a transcriptional activator that functions downstream of the highly conserved Receptor Tyrosine Kinase (RTK) signaling pathway. Pnt is an ETS family transcription factor and encodes for two proteins, PntP1 and PntP2. However, while PntP1 is constitutively active, PntP2 is only active after being phosphorylated by MAPK in the RTK pathway. As mutations in pnt perturb the development of several tissues, we wanted to examine the effect and efficacy of using RNAi to target Pnt. We have expressed pnt RNAi in the eyes, oocyte, and heart cells using three different RNAi lines: Valium20, Valium10, and VDRC. Valium20 is distinct since it generates a short hairpin RNA (shRNA), while Valium10 and VDRC produce long hairpin dsRNA. We found that for each tissue examined Valium20 exhibited the strongest phenotype while the Valium10 and VDRC lines produced varying levels of severity and that the long hairpin RNA produced by the Valium10 and VDRC lines are unable to effectively knockdown pnt in embryonic tissues.

The relationship between long-range chromatin occupancy and polymerization of the Drosophila ETS family transcriptional repressor Yan.

ETS family transcription factors are evolutionarily conserved downstream effectors of Ras/MAPK signaling with critical roles in development and cancer. In Drosophila, the ETS repressor Yan regulates cell proliferation and differentiation in a variety of tissues; however, the mechanisms of Yan-mediated repression are not well understood and only a few direct target genes have been identified. Yan, like its human ortholog TEL1, self-associates through an N-terminal sterile α-motif (SAM), leading to speculation that Yan/TEL1 polymers may spread along chromatin to form large repressive domains. To test this hypothesis, we created a monomeric form of Yan by recombineering a point mutation that blocks SAM-mediated self-association into the yan genomic locus and compared its genome-wide chromatin occupancy profile to that of endogenous wild-type Yan. Consistent with the spreading model predictions, wild-type Yan-bound regions span multiple kilobases. Extended occupancy patterns appear most prominent at genes encoding crucial developmental regulators and signaling molecules and are highly conserved between Drosophila melanogaster and D. virilis, suggesting functional relevance. Surprisingly, although occupancy is reduced, the Yan monomer still makes extensive multikilobase contacts with chromatin, with an overall pattern similar to that of wild-type Yan. Despite its near-normal chromatin recruitment, the repressive function of the Yan monomer is significantly impaired, as evidenced by elevated target gene expression and failure to rescue a yan null mutation. Together our data argue that SAM-mediated polymerization contributes to the functional output of the active Yan repressive complexes that assemble across extended stretches of chromatin, but does not directly mediate recruitment to DNA or chromatin spreading.

The SAM domain of human TEL2 can abrogate transcriptional output from TEL1 (ETV-6) and ETS1/ETS2.

Regulation of gene expression downstream of the Receptor Tyrosine Kinase signaling pathway in Drosophila relies on a transcriptional effector network featuring two conserved Ets family proteins, Yan and Pointed, known as TEL1 (ETV6) and ETS1/ETS2, respectively, in mammals. As in Drosophila, both TEL1 and ETS1/ETS2 operate as Ras pathway transcriptional effectors and misregulated activity of either factor has been implicated in many human leukemias and solid tumors. Providing essential regulation to the Drosophila network, direct interactions with the SAM domain protein Mae attenuate both Yan-mediated repression and PointedP2-mediated transcriptional activation. Given the critical contributions of Mae to the Drosophila circuitry, we investigated whether the human Ets factors TEL1 and ETS1/ETS2 could be subject to analogous regulation. Here we demonstrate that the SAM domain of human TEL2 can inhibit the transcriptional activities of ETS1/2 and TEL1. Drosophila Mae can also attenuate human ETS1/ETS2 function, suggesting there could be cross-species conservation of underlying mechanism. In contrast, Mae is not an effective inhibitor of TEL1, suggesting the mode of TEL2SAM-mediated inhibition of TEL1 may be distinct from how Drosophila Mae antagonizes Yan. Together our results reveal both further similarities and new differences between the mammalian and Drosophila networks and more broadly suggest that SAM domain-mediated interactions could provide an effective mechanism for modulating output from the TEL1 and ETS1/2 oncogenes.

Sterile alpha motif domain-mediated self-association plays an essential role in modulating the activity of the Drosophila ETS family transcriptional repressor Yan.

The ETS family transcriptional repressor Yan is an important downstream target and effector of the receptor tyrosine kinase (RTK) signaling pathway in Drosophila melanogaster. Structural and biochemical studies have shown that the N-terminal sterile alpha motif (SAM) of Yan is able to self associate to form a helical polymeric structure in vitro, although the extent and functional significance of self-association of full-length Yan remain unclear. In this study, we demonstrated that full-length Yan self associates via its SAM domain to form higher-order complexes in living cells. Introduction of SAM domain missense mutations that restrict Yan to a monomeric state reduces Yan's transcriptional repression activity and impairs its function during embryonic and retinal development. Coexpression of combinations of SAM domain mutations that permit the formation of Yan dimers, but not higher-order oligomers, increases activity relative to that of monomeric Yan, but not to the level obtained with wild-type Yan. Mechanistically, self-association directly promotes transcriptional repression of target genes independent of its role in limiting mitogen-activated protein kinase (MAPK)-mediated phosphorylation and nuclear export of Yan. Thus, we propose that the formation of higher-order Yan oligomers contributes to proper repression of target gene expression and RTK signaling output in developing tissues.

Intersection of signal transduction pathways and development.

One of the challenges of modern biology is to understand how cells within a developing organism generate, integrate, and respond to dynamic informational cues. Based on over two decades of intensive research, many parts and subroutines of the responsible signal transduction networks have been identified and functionally characterized. From this work, it has become evident that a complicated interplay between signaling pathways, involving extensive feedback regulation and multiple levels of cross-talk, underlies even the "simplest" developmental decision. Thus a signaling pathway can no longer be thought of as a rigid linear process, but rather must be considered a dynamic, self-interacting, and self-adjusting network. The Epidermal Growth Factor Receptor tyrosine kinase signaling pathway provides a prime vantage point from which to explore emerging principles in developmental signal transduction.

MAE, a dual regulator of the EGFR signaling pathway, is a target of the Ets transcription factors PNT and YAN.

Ets transcription factors play crucial roles in regulating diverse cellular processes including cell proliferation, differentiation and survival. Coordinated regulation of the Drosophila Ets transcription factors YAN and POINTED is required for eliciting appropriate responses to Receptor Tyrosine Kinase (RTK) signaling. YAN, a transcriptional repressor, and POINTED, a transcriptional activator, compete for regulatory regions of common target genes, with the ultimate outcome likely influenced by context-specific interactions with binding partners such as MAE. Previous work in cultured cells has led us to propose that MAE attenuates the transcriptional activity of both YAN and POINTED, although its effects on POINTED remain controversial. Here we describe a new layer of complexity to this regulatory hierarchy whereby mae expression is itself directly regulated by the opposing action of YAN and POINTED. In addition, we report that MAE can antagonize POINTED function during eye development; a finding that suggests MAE operates as a dual positive and negative regulator of RTK-mediated signaling in vivo. Together our results lead us to propose that a combination of protein-protein and transcriptional interactions between MAE, YAN and POINTED establishes a complex regulatory circuit that ensures that both down-regulation and activation of the RTK pathway occur appropriately according to specific developmental context.