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Inhibition of Tumor Necrosis Factor- through Selective Blockade of Pre-mRNA Splicing by Shikonin

Graduate Institute of Life Sciences, National Defense Medical Center (S.-C.C., N.-S.Y.); and Agricultural Biotechnology Research Center, Academia Sinica (S.-C.C., N.-S.Y.), Taipei, Taiwan, Republic of China

We previously developed a gene-gun-based in vivo screening system and identified shikonin as a potent suppressor of tumor necrosis factor- (TNF-) gene expression. Here, we show that shikonin selectively inhibits the expression of TNF- 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-, 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- 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- pre-mRNA splicing may constitute a promising target for future anti-inflammatory application.

Address correspondence to: Dr. Ning-Sun Yang, Agricultural Biotechnology Research Center, Academia Sinica, 11529, Taipei, Taiwan, ROC. E-mail: nsyang{at}gate.sinica.edu.tw