TFR2 BETA Isoform Is Differentially Produced Compared to ALFA and Has Specific Function at Young Age.

The transferrin receptor 2 gene (TFR2) encodes for a transmembrane protein that plays a pivotal role in iron metabolism, since TFR2 mutations are responsible of type 3 Hereditary Hemochromatosis (HH).

Together with other HH proteins (Hfe and Rgmc) Tfr2 participates to modulation of Hepcidin (Hepc) production, a small peptide able to regulate body iron availability through its negative effect on cellular iron exporter Ferroportin (Fpn1).

Tfr2 protein is a member of the transferrin receptor family, showing moderate homology to transferrin receptor (Tfrc). It is able to bind transferrin even thought at lower affinity compared to TFRC. Two alternative transcripts of TFR2 have been reported, named alfa and beta. TFR2 alfa mRNA is highly expressed in the hepatocytes while TFR2 beta has a low ubiquitous expression (Kawabata et al, 1999).

The beta isoform is identical to alfa but it lacks the transmembrane and cytoplasmic domains; it has been considered an alternative splicing form and its role is still poorly understood. Nevertheless, clinical observations support the hypothesis that TFR2 beta has an important functional role since HFE3 patients with mutations not compromising its production have a milder phenotype compared to those unable to synthesize both alfa and beta TFR2 isoforms (Roetto et al,2002; Le Gac et al, 2004).

In order to elucidate whether TFR2 beta was differentially expressed and produced during life span its mRNA and translated protein were analyzed in C57BL/6 mice during their growth and adult age.

Since Tfr2 beta protein has never been demonstrated to exist before, its cDNA has been cloned in an expression vector and transfected in HEK293T cells.

Cells lysates have been electhrophoresed, transferred onto nitrocellulose membrane, decorated with Tfr2 Ab (Santa Cruz Biotechnoloy, Inc) and visualized on Western Blot. A 65 kDa band has been obtained from the transfection, consistent with the aminoacid sequence predicted molecular weight, and used as a positive control for further in vivo experiments. C57BL/6 mice livers from seven different ages, starting from birth until adult age (post natal days 0, 1, 3, 7, 14, 28, 40 and 60) have been analyzed in WB utilizing Tfr2 antibody. At least three mice per point have been analyzed. One band of a molecular weight of approximately 95 KDa has been detected during whole animal life, quantitatively increasing during life time, corresponding to Tfr2 alfa. Meanwhile, the beta isoform appeared to be consistently produced until the 7th day after birth and disappears starting from day 14.

To evaluate the possible consequences of this result on iron metabolism, HH proteins (Hfe, Fpn1, Rgmc) mRNA expression pattern has been analyzed in real time PCR at the same points, as well as Hepcidin (Hepc) transcript, in order to evaluate whether TFR2 beta production could in some way influence Hepc modulation.

HEPC transcription resulted to be significantly downmodulated from day 0 after birth until day 14 as compared to later stages in mice life (adulthood).

HFE transcription resulted to be lower in young animals compared to adults while FPN1 increased significantly between day 3 and 7, according to HEPC decreasing.

On the whole, these results suggest a functional role for the Tfr2 beta isoform in iron sensing, since this isoform resulted to be selectively produced during animal growth. At the same life period Hepc transcription resulted to be significantly downmodulated, allowing in this way increased iron absorption. These data are consistent with an higher iron requirement necessary for massive animal body growth.