Differential HFE Gene Expression Is Regulated by Alternative Splicing in Human Tissues

Abstract

BackgroundThe pathophysiology of HFE-derived Hereditary Hemochromatosis and the function of HFE protein in iron homeostasis remain uncertain. Also, the role of alternative splicing in HFE gene expression regulation and the possible function of the corresponding protein isoforms are still unknown. The aim of this study was to gain insights into the physiological significance of these alternative HFE variants.Methodology/Principal FindingsAlternatively spliced HFE transcripts in diverse human tissues were identified by RT-PCR, cloning and sequencing. Total HFE transcripts, as well as two alternative splicing transcripts were quantified using a real-time PCR methodology. Intracellular localization, trafficking and protein association of GFP-tagged HFE protein variants were analysed in transiently transfected HepG2 cells by immunoprecipitation and immunofluorescence assays.Alternatively spliced HFE transcripts present both level- and tissue-specificity. Concerning the exon 2 skipping and intron 4 inclusion transcripts, the liver presents the lowest relative level, while duodenum presents one of the highest amounts. The protein resulting from exon 2 skipping transcript is unable to associate with β2M and TfR1 and reveals an ER retention. Conversely, the intron 4 inclusion transcript gives rise to a truncated, soluble protein (sHFE) that is mostly secreted by cells to the medium in association with β2M.Conclusions/Significance HFE gene post-transcriptional regulation is clearly affected by a tissue-dependent alternative splicing mechanism. Among the corresponding proteins, a sHFE isoform stands out, which upon being secreted into the bloodstream, may act in remote tissues. It could be either an agonist or antagonist of the full length HFE, through hepcidin expression regulation in the liver or by controlling dietary iron absorption in the duodenum.

Preliminary Investigation of Bottlenose Dolphins (Tursiops truncatus) forhfeGene–related Hemochromatosis

“…Seven alternative transcripts in addition to the hfe wild type are expressed by various human tissues, particularly the liver and duodenum. These alternative transcripts contain exon deletions and intron insertions and are suggested to result in different functional proteins (Martins et al 2011). Thus the alternative splicing in our samples may represent a normal process within the dolphin liver.…”

“…Hepatocytes produce HFE, which is involved in regulation of iron absorption and homeostasis. HFE is a major histocompatibility complex (MHC) class 1-like protein that interacts with transferrin receptor 2 (TfR2) on hepatocyte cell surfaces (Martins et al 2011). Increased plasma iron levels are sensed by the HFE-TfR2 complex, upregulating hepcidin expression.…”

The soluble form of HFE protein regulates hephaestin mRNA expression in the duodenum through an endocytosis-dependent mechanism

“…The expression of HFE undergoes a complex post-transcriptional regulation that involves alternative polyadenylation and splicing mechanisms. So far, there were identified four different polyadenylation signals and more than ten transcripts resulting from HFE alternative splicing [11][12][13]. The expression of these variants seems to be ubiquitous and their functional characterization is not yet complete.…”

“…These alternative transcripts result from the total inclusion of HFE intron 4 or the inclusion of the first 66 base-pairs of intron 4 and, in both cases, an in frame premature termination codon is formed, six nucleotides from the exon 4/intron 4 boundary [12]. The sHFE protein preserves an intact α3 domain but lacks the transmembrane domain and the cytoplasmic tail.…”

“…The sHFE protein preserves an intact α3 domain but lacks the transmembrane domain and the cytoplasmic tail. Consequently, it maintains the ability to associate with β 2 M but not with TfR1 and it has been shown to be secreted to the extracellular environment in several cell types [12]. However, the function of sHFE is still unknown.…”