Received for publication November 27, 2006.
Revised March 12, 2007.
Accepted for publication March 12, 2007.

Inhibition of TNF- through Selective Blockade of Pre-mRNA Splicing by Shikonin

Abstract

We previously developed a gene-gun-based in vivo screening system and identified shikonin as a potent suppressor of tumor necrosis factor-alpha (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, interleukine-8 (IL-8), was affected. Moreover, co-treatment with LPS and shikonin increased the end-point protein production of IL-8, accompanied by suppressed activation of the dsRNA-activated protein kinase (PKR) pathway. Because PKR inactivation has been shown to downregulate 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.

Key words: Tumor necrosis factor, Protein Kinases (other), Regulation - post-transcriptional