an-ecdysone-receptor-based-singular-gene-switch-for-deliberate

An Ecdysone Receptor-based Singular Gene Switch for Deliberate Expression of Transgene with Robustness, Reversibility, and Negligible Leakiness

Precise control of transgene expression is desirable in biological and clinical studies. However, because the binary feature of currently employed gene ...

Chungnam National University

Controlled gene expression is an indispensable technique in biomedical research. Here, we report a convenient, straightforward, and reliable way to induce expression of a gene of interest with negligible background expression compared to the most widely used tetracycline (Tet)-regulated system. Exploiting a Drosophila ecdysone receptor (EcR)-based gene regulatory system, we generated nonviral and adenoviral singular vectors designated as pEUI(+) and pENTR-EUI, respectively, which contain all the required elements to guarantee regulated transgene expression (GAL4-miniVP16-EcR, termed GvEcR hereafter, and 10 tandem repeats of an upstream activation sequence promoter followed by a multiple cloning site). Through the transient and stable transfection of mammalian cell lines with reporter genes, we validated that tebufenozide, an ecdysone agonist, reversibly induced gene expression, in a dose- and time-dependent manner, with negligible background expression. In addition, we created an adenovirus derived from the pENTR-EUI vector that readily infected not only cultured cells but also rodent tissues and was sensitive to tebufenozide treatment for regulated transgene expression. These results suggest that EcR-based singular gene regulatory switches would be convenient tools for the induction of gene expression in cells and tissues in a tightly controlled fashion. 

Ecdysone Receptor-based Singular Gene Switches for Regulated Transgene Expression in Cells and Adult Rodent Tissues.

Pseudolysimachion rotundum var. subintegrum is utilized as a traditional herbal remedy to treat cough, bronchitis, and asthma in Korea, Russia, China, and Europe. Here, we show that 3-methoxy-catalposide, a novel iridoide glycoside isolated from P. rotundum var. subintegrum has the anti-inflammatory activity in lipopolysaccharide (LPS)-stimulated macrophages. The chemical structure of 3-methoxy-catalposide was determined by NMR, optical rotation and HRESIMS. In in vitro experiment, RAW264.7 cells were treated with 3-methoxy-catalposide for 2h before exposure to LPS for different times. Inflammatory gene and protein expressions were assayed using RT-PCR and ELISA. Activities of signal proteins were examined using western analysis. Our results demonstrated that 3-methoxy-catalposide significantly inhibits the expression of cyclooxygenase (COX)-2 and inducible nitric oxide synthase (iNOS) in RAW264.7 cells stimulated by LPS, thereby suppressing the release of prostaglandin E2 (PGE) and nitric oxide (NO). Moreover, 3-methoxy-catalposide markedly reduced the LPS-induced expression of pro-inflammatory genes, such as interleukin (IL)-6, IL-1β, and TNF-α. Further, 3-methoxy-catalposide inhibited both LPS-induced activation of three MAP kinases (ERK 1/2, JNK, and p38) and the nuclear translocation of NF-κB and AP-1. These results support that 3-methoxy-catalposide may be a promising candidate for inflammation treatment.

3-Methoxy-catalposide inhibits inflammatory effects in lipopolysaccharide-stimulated RAW264.7 macrophages.

Fisetin (3,7,3',4'-tetrahydroxyflavone), a natural flavonoid, is a therapeutic agent for respiratory inflammatory diseases such as chronic obstructive pulmonary disease (COPD). However, detailed molecular mechanisms regarding the target protein of fisetin remain unknown. Fisetin significantly reduces tumour necrosis factor alpha (TNF-α)-induced interleukin (IL)-8 levels by inhibiting both nuclear factor kappa B (NF-κB) transcriptional activity and the phosphorylation of its upstream effectors. We show that fisetin prevents interactions between protein kinase C (PKC)δ and TNF receptor-associated factor 2 (TRAF2), thereby inhibiting the inhibitor of kappa B kinase (IKK)/NF-κB downstream signalling cascade. Furthermore, we found that fisetin directly binds to PKCδ in vitro. Our findings provide evidence that fisetin inhibits the TNF-α-activated IKK/NF-κB cascade by targeting PKCδ, thereby mediating inflammatory diseases such as COPD. These data suggest that fisetin is a good therapeutic drug for the treatment of inflammatory lung diseases, such as COPD, by inhibiting the TNF-α/NF-κB signalling pathway.

Fisetin inhibits TNF-α/NF-κB-induced IL-8 expression by targeting PKCδ in human airway epithelial cells.

Piscroside C, isolated from Pseudolysimachion rotundum var. subintegrum, is a novel iridoid glycoside with therapeutic efficacy in a mouse model of chronic obstructive pulmonary disease (COPD). Piscroside C has been reported as a constituent of YPL-001 (under Phase 2a study, ClinicalTrials.gov identifier NCT02272634).

Article	Year
Ecdysone Receptor-based Singular Gene Switches for Regulated Transgene Expression in Cells and Adult Rodent Tissues. Molecular therapy. Nucleic acids\| PubMed ID: 27673563	2016
3-Methoxy-catalposide inhibits inflammatory effects in lipopolysaccharide-stimulated RAW264.7 macrophages. Cytokine\| PubMed ID: 28011397	2017
Fisetin inhibits TNF-α/NF-κB-induced IL-8 expression by targeting PKCδ in human airway epithelial cells. Cytokine\| PubMed ID: 29396047	2018
Piscroside C inhibits TNF-α/NF-κB pathway by the suppression of PKCδ activity for TNF-RSC formation in human airway epithelial cells. Phytomedicine : international journal of phytotherapy and phytopharmacology\| PubMed ID: 29496167	2018

Seoghyun Lee

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