Unbiased RNAi screen for hepcidin regulators links hepcidin suppression to proliferative Ras/RAF and nutrient-dependent mTOR signaling.

Abstract

The hepatic hormone hepcidin is a key regulator of systemic iron metabolism. Its expression is largely regulated by 2 signaling pathways: the "iron-regulated" bone morphogenetic protein (BMP) and the inflammatory JAK-STAT pathways. To obtain broader insights into cellular processes that modulate hepcidin transcription and to provide a resource to identify novel genetic modifiers of systemic iron homeostasis, we designed an RNA interference (RNAi) screen that monitors hepcidin promoter activity after the knockdown of 19 599 genes in hepatocarcinoma cells. Interestingly, many of the putative hepcidin activators play roles in signal transduction, inflammation, or transcription, and affect hepcidin transcription through BMP-responsive elements. Furthermore, our work sheds light on new components of the transcriptional machinery that maintain steady-state levels of hepcidin expression and its responses to the BMP- and interleukin-6-triggered signals. Notably, we discover hepcidin suppression mediated via components of Ras/RAF MAPK and mTOR signaling, linking hepcidin transcriptional control to the pathways that respond to mitogen stimulation and nutrient status. Thus using a combination of RNAi screening, reverse phase protein arrays, and small molecules testing, we identify links between the control of systemic iron homeostasis and critical liver processes such as regeneration, response to injury, carcinogenesis, and nutrient metabolism.

• Authors: Katarzyna Mleczko-Sanecka, Franziska Roche, Ana Rita da Silva, Debora Call, Flavia D'Alessio, Anan Ragab, Philip E Lapinski, Ramesh Ummanni, Ulrike Korf, Christopher Oakes, Georg Damm, Lorenza A D'Alessandro, Ursula Klingmüller, Philip D King, Michael Boutros, Matthias W Hentze, Martina U Muckenthaler
• Journal: Blood (Blood) Vol. 123 Issue 10 Pg. 1574-85 (Mar 06 2014) ISSN: 1528-0020 [Electronic] United States
• PMID: 24385536 (Publication Type: Journal Article, Research Support, N.I.H., Extramural, Research Support, Non-U.S. Gov't)

Chemical References

• Bone Morphogenetic Proteins
• Hepcidins
• Proto-Oncogene Proteins c-raf
• TOR Serine-Threonine Kinases
• Mitogen-Activated Protein Kinases
• Proto-Oncogene Proteins p21(ras)

Topics

• Animals
• Bone Morphogenetic Proteins (metabolism)
• Cell Line
• Gene Expression Profiling
• Gene Expression Regulation
• Hepcidins (genetics, metabolism)
• Humans
• Male
• Mice
• Mice, Knockout
• Mitogen-Activated Protein Kinases (metabolism)
• Protein Binding
• Proto-Oncogene Proteins c-raf (metabolism)
• Proto-Oncogene Proteins p21(ras) (metabolism)
• RNA Interference
• Reproducibility of Results
• Response Elements
• Signal Transduction
• TOR Serine-Threonine Kinases (metabolism)
• Transcription, Genetic