Assessment of Anti-TNF-α Activities in Keratinocytes Expressing Inducible TNF- α: A Novel Tool for Anti-TNF-α Drug Screening.
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...
| Main Authors: | , , , |
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| Format: | Article |
| Language: | English |
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Public Library of Science (PLoS)
2016-01-01
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| Series: | PLoS ONE |
| Online Access: | http://europepmc.org/articles/PMC4945017?pdf=render |
| _version_ | 1811335875645145088 |
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| author | Sutthirat Udommethaporn Tewin Tencomnao Eileen M McGowan Viroj Boonyaratanakornkit |
| author_facet | Sutthirat Udommethaporn Tewin Tencomnao Eileen M McGowan Viroj Boonyaratanakornkit |
| author_sort | Sutthirat Udommethaporn |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-04-13T17:31:17Z |
| format | Article |
| id | doaj.art-2ef6a14ef75344e8b9bbeee8d77c416d |
| institution | Directory Open Access Journal |
| issn | 1932-6203 |
| language | English |
| last_indexed | 2024-04-13T17:31:17Z |
| publishDate | 2016-01-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS ONE |
| spelling | doaj.art-2ef6a14ef75344e8b9bbeee8d77c416d2022-12-22T02:37:33ZengPublic Library of Science (PLoS)PLoS ONE1932-62032016-01-01117e015915110.1371/journal.pone.0159151Assessment of Anti-TNF-α Activities in Keratinocytes Expressing Inducible TNF- α: A Novel Tool for Anti-TNF-α Drug Screening.Sutthirat UdommethapornTewin TencomnaoEileen M McGowanViroj BoonyaratanakornkitTumor 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.http://europepmc.org/articles/PMC4945017?pdf=render |
| spellingShingle | Sutthirat Udommethaporn Tewin Tencomnao Eileen M McGowan Viroj Boonyaratanakornkit Assessment of Anti-TNF-α Activities in Keratinocytes Expressing Inducible TNF- α: A Novel Tool for Anti-TNF-α Drug Screening. PLoS ONE |
| title | Assessment of Anti-TNF-α Activities in Keratinocytes Expressing Inducible TNF- α: A Novel Tool for Anti-TNF-α Drug Screening. |
| title_full | Assessment of Anti-TNF-α Activities in Keratinocytes Expressing Inducible TNF- α: A Novel Tool for Anti-TNF-α Drug Screening. |
| title_fullStr | Assessment of Anti-TNF-α Activities in Keratinocytes Expressing Inducible TNF- α: A Novel Tool for Anti-TNF-α Drug Screening. |
| title_full_unstemmed | Assessment of Anti-TNF-α Activities in Keratinocytes Expressing Inducible TNF- α: A Novel Tool for Anti-TNF-α Drug Screening. |
| title_short | Assessment of Anti-TNF-α Activities in Keratinocytes Expressing Inducible TNF- α: A Novel Tool for Anti-TNF-α Drug Screening. |
| title_sort | assessment of anti tnf α activities in keratinocytes expressing inducible tnf α a novel tool for anti tnf α drug screening |
| url | http://europepmc.org/articles/PMC4945017?pdf=render |
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