Abstract
Interleukin-6 (IL-6) is a proinflammatory cytokine that exerts a wide range of cellular, physiological, and pathophysiological responses. Pyrrolidine dithiocarbamate (PDTC) antagonizes the cellular responsiveness to IL-6 through impairment in signal transducer and activator of transcription-3 activation and downstream signaling. To further elucidate the biological properties of PDTC, global gene expression profiling of human HepG2 hepatocellular carcinoma cells was carried out after treatment with PDTC or IL-6 for up to 8 h. Through an unbiased pathway analysis method, gene array analysis showed dramatic and temporal differences in expression changes in response to PDTC versus IL-6. A significant number of genes associated with metabolic pathways, inflammation, translation, and mitochondrial function were changed, with ribosomal protein genes and DNA damage-inducible transcript 4 protein (DDIT4) primarily up-regulated with PDTC but down-regulated with IL-6. Quantitative polymerase chain reaction and Western blot analyses validated the microarray data and showed the reciprocal expression pattern of the mammalian target of rapamycin (mTOR)-negative regulator DDIT4 in response to PDTC versus IL-6. Cell treatment with PDTC resulted in a rapid and sustained activation of Akt and subsequently blocked the IL-6-mediated increase in mTOR complex 1 function through up-regulation in DDIT4 expression. Conversely, down-regulation of DDIT4 with small interfering RNA dampened the capacity of PDTC to block IL-6-dependent mTOR activation. The overall protein biosynthetic capacity of the cells was severely blunted by IL-6 but increased in a rapamycin-independent pathway by PDTC. These results demonstrate a critical effect of PDTC on mTOR complex 1 function and provide evidence that PDTC can reverse IL-6-related signaling via induction of DDIT4.
Footnotes
This research was supported by the Intramural Research Program of the National Institutes of Health, National Institute on Aging.
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
doi:10.1124/jpet.111.185678.
↵ The online version of this article (available at http://jpet.aspetjournals.org) contains supplemental material.
-
ABBREVIATIONS:
- PDTC
- pyrrolidine dithiocarbamate
- HSF1
- heat shock factor 1
- IL-6
- interleukin-6
- STAT3
- signal transducer and activator of transcription-3
- mTOR
- mammalian target of rapamycin
- mTORC
- mTOR complex
- TSC
- tuberous sclerosis complex
- IRES
- internal ribosome entry site
- DDIT4
- DNA damage-inducible transcript 4 protein
- PCR
- polymerase chain reaction
- siRNA
- small interfering RNA
- GSK
- glycogen synthase kinase.
- Received July 4, 2011.
- Accepted September 12, 2011.
- U.S. Government work not protected by U.S. copyright
JPET articles become freely available 12 months after publication, and remain freely available for 5 years.Non-open access articles that fall outside this five year window are available only to institutional subscribers and current ASPET members, or through the article purchase feature at the bottom of the page.
|