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|Title: ||A soluble HFE splice variant seems to regulate the expression of duodenal cytochrome b and hephaestin contributing to iron metabolism regulation|
|Authors: ||Silva, Bruno|
|Issue Date: ||Nov-2012|
|Publisher: ||Instituto Nacional de Saúde Doutor Ricardo Jorge, IP|
|Abstract: ||INTRODUCTION: Hereditary Hemochromatosis is an autosomal recessive disorder characterized by excessive intestinal iron absorption and pathological iron deposition in organs such as liver, heart and pancreas. The disease is predominantly caused by homozygosity for the p.C282Y mutation in HFE, which impairs protein association with its chaperone beta-2 microglobulin (B2M), for correct folding and traffic to the cell surface. Several alternative HFE transcripts have been reported but their functional significance remains elusive. Since we have identified an alternative HFE transcript due to intron 4 inclusion, we aimed to investigate its physiological role on iron homeostasis.
MATERIALS AND METHODS: We analysed the expression level of the alternative transcript in several human tissues by quantitative Real-Time PCR. In addition, we produced the corresponding GFP-tagged HFE protein variant. HepG2 cells were transfected with this construct and protein cellular location analyzed by immunofluorescence using several antibodies. In parallel, immunoprecipitation was performed. Finally, the variant was over-expressed in a duodenal cell line (Hutu-80) under normal and iron overload conditions and the expression of several iron metabolism related genes (TFR1, DMT1, CYBRD1, SLC40A1 and HEPH) was evaluated by quantitative Real-Time PCR.
RESULTS: We have found that the HFE_intron 4 inclusion transcript has an ubiquitous expression, being its relative expression higher in duodenum and lower in the liver. Also, we found that it gives rise to a truncated soluble protein (sHFE) that is secreted by cells maintaining its interaction with B2M. Its overexpression in HuTu-80 cells revealed that the sHFE is able to down-regulate the duodenal cytochrome b (CYBRD1) expression, as it happens with the HFE_full length protein. Also, it seems to play a specific role in the regulation of hephaestin (HEPH) expression.
CONCLUSIONS: Through this study we might have unveiled the contribution of the sHFE variant to iron homeostasis. In fact, sHFE may be secreted into the bloodstream and act in remote tissues such as duodenum, down-regulating the expression of some of the iron metabolism related genes, as CYBRD1 and HEPH, and consequently reducing dietary iron absorption, preventing iron overload and contributing to iron metabolism regulation.
Partially funded by FCT: PTDC/SAU-GMG/64494/2006; CIGMH; SFRH/BD/21340/2005 and SFRH/BD/60718/2009.
The authors declare no competing interests.|
|Peer Reviewed: ||yes|
|Appears in Collections:||DGH - Posters/abstracts em congressos nacionais|
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