Abstract
We previously developed a gene-gun-based in vivo screening system and identified shikonin as a potent suppressor of tumor necrosis factor-α (TNF-α) gene expression. Here, we show that shikonin selectively inhibits the expression of TNF-α at the RNA splicing level. Treatment of lipopolysaccharide-stimulated human primary monocytes and THP-1 cells with shikonin resulted in normal transcriptional induction of TNF-α, but unspliced pre-mRNA accumulated at the expense of functional mRNA. This effect occurred with noncytotoxic doses of shikonin and was highly specific, because mRNA production of neither a housekeeping gene nor another inflammatory cytokine gene, interleukin-8 (IL-8), was affected. Moreover, cotreatment with lipopolysaccharide (LPS) and shikonin increased the endpoint protein production of IL-8, accompanied by suppressed activation of the double-stranded RNA-activated protein kinase (PKR) pathway. Because PKR inactivation has been shown to down-regulate the splicing process of TNF-α RNA and interfere with translation, our findings suggest that shikonin may achieve differential modulation of cytokine protein expression through inactivation of the PKR pathway and reveal that regulation of TNF-α pre-mRNA splicing may constitute a promising target for future anti-inflammatory application.
Footnotes
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This work was supported by grant 94F002-1 from Academia Sinica, Taipei, Taiwan, Republic of China.
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Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
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doi:10.1124/mol.106.032821.
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ABBREVIATIONS: TNF-α, tumor necrosis factor-α; PKR, double-stranded RNA-activated protein kinase; 2-AP, 2-aminopurine; CHX, cycloheximide; LPS, lipopolysaccharide; SB203580, 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole; U0126, 1,4-diamino-2,3-dicyano-1,4-bis(methylthio)butadiene; RT-PCR, reverse transcriptase-polymerase chain reaction; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ELISA, enzyme-linked immunosorbent assay; PBST, phosphate-buffered saline/Tween 20; IL, interleukin; 2APRE, 2-AP responsive element; eIF2α, eukaryotic initiation factor-2α.
- Received November 21, 2006.
- Accepted March 12, 2007.
- The American Society for Pharmacology and Experimental Therapeutics
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