title: Assessment of Anti-TNF-α Activities in Keratinocytes Expressing Inducible TNF- α: A Novel Tool for Anti-TNF-α Drug Screening.
abstract: Tumor necrosis factor alpha (TNF-α) is a pro-inflammatory cytokine important in normal and pathological biological processes. Newly synthesized pro-TNF-α is expressed on the plasma membrane and cleaved to release soluble TNF-α protein: both are biologically active. Secreted TNF-α signals through TNF receptors and the membrane-bound TNF-α acts by cell contact-dependent signaling. Anti-TNF-α antibodies have been used effectively for treatment of chronic inflammation, however with adverse side effects. Thus, there is a need for new anti-TNF-α small molecule compounds. Anti-TNF-α activity assays involve treatment of keratinocytes with exogenous TNF-α before or after anti-TNF-α incubation. However, this model fails to address the dual signaling of TNF-α. Here we describe a Doxycycline (Dox)-inducible TNF-α (HaCaT-TNF-α) expression system in keratinocytes. Using this in-vitro model, we show cell inhibition and induced expression of pro-inflammatory cytokines and markers, including IL-1β, IL-6, IL-8, NF-κB1, and KRT-16, similar to cells treated with exogenous TNF-α. Sufficient secreted TNF-α produced also activated IL-1β and IL-8 expression in wt HaCaT cells. Importantly, stimulated expression of IL-1β and IL-8 in HaCaT-TNF-α were blocked by Quercetin, a flavanol shown to possess anti-TNF-α activities. This novel in vitro cell model provides an efficient tool to investigate the dual signaling of TNF-α. Importantly, this model provides an effective, fast, and simple screening for compounds with anti-TNF-α activities for chronic inflammatory disease therapies.
authors.affiliation: Graduate Program in Clinical Biochemistry and Molecular Medicine, Faculty of Allied Health Sciences, Chulalongkorn University, Bangkok 10330, Thailand.
authors.forename: Sutthirat
authors.initials: S
authors.lastname: Udommethaporn
authors.affiliation: Graduate Program in Clinical Biochemistry and Molecular Medicine, Faculty of Allied Health Sciences, Chulalongkorn University, Bangkok 10330, Thailand.|Department of Clinical Chemistry, Faculty of Allied Health Sciences, Chulalongkorn University, Bangkok 10330, Thailand.
authors.forename: Tewin
authors.initials: T
authors.lastname: Tencomnao
authors.affiliation: Chronic Disease Solutions Team, School of Life Sciences, University of Technology Sydney, Ultimo, 2007, Sydney, Australia.
authors.forename: Eileen M
authors.initials: EM
authors.lastname: McGowan
authors.affiliation: Graduate Program in Clinical Biochemistry and Molecular Medicine, Faculty of Allied Health Sciences, Chulalongkorn University, Bangkok 10330, Thailand.|Department of Clinical Chemistry, Faculty of Allied Health Sciences, Chulalongkorn University, Bangkok 10330, Thailand.
authors.forename: Viroj
authors.identifier: http://orcid.org/0000-0003-1800-7836
authors.initials: V
authors.lastname: Boonyaratanakornkit
mesh_terms: D000893:Anti-Inflammatory Agents / Q000494:pharmacology; D000911:Antibodies, Monoclonal; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D016207:Cytokines / Q000235:genetics; D004318:Doxycycline / Q000494:pharmacology; D004353:Drug Evaluation, Preclinical / Q000379:methods; D015870:Gene Expression / Q000187:drug effects; D057809:HEK293 Cells; D006801:Humans; D018836:Inflammation Mediators / Q000378:metabolism; D015603:Keratinocytes / Q000187:drug effects / Q000276:immunology* / Q000378:metabolism*; D018448:Models, Immunological; D015398:Signal Transduction / Q000187:drug effects; D014409:Tumor Necrosis Factor-alpha / Q000037:antagonists & inhibitors* / Q000276:immunology / Q000378:metabolism*
chemical_list: D000893:Anti-Inflammatory Agents; D000911:Antibodies, Monoclonal; D016207:Cytokines; D018836:Inflammation Mediators; D014409:Tumor Necrosis Factor-alpha; D004318:Doxycycline