Biochemical and Biophysical Research Communications
Knockdown of β2-microglobulin perturbs the subcellular distribution of HFE and hepcidin
Section snippets
Materials and methods
Plasmid constructs, cell lines, plasmid transfection, and RNAi. The HA-HFE construct was a gift from Kostas Pantopoulos [8]. HuTu 80 cells (duodenal adenocarcinoma cells) were purchased from ATCC. Cells were grown, transfected and analysed by immunofluorescence microscopy as described previously [9], [10]. β2M expression in HuTu 80 cells was depleted by transfection with siGEMONE SMARTpool reagents directed against β2M (Dharmacon). As a control, HuTu 80 cells were treated with siCONTROL
Knockdown of β2M restricts HFE localisation to the endoplasmic reticulum
In an attempt to gain a better understanding of the cellular role of β2M, we depleted β2M expression in HuTu 80 cells by RNAi and investigated the effects its loss has on the subcellular distribution of key proteins implicated in iron homeostasis. We found that treatment of HuTu 80 cells with β2M siRNA substantially depleted β2M levels as assessed by confocal immunofluorescence microscopy (Fig. 1A). As previously reported [11], we found that in cells exogenously expressing HFE, HFE displayed a
Discussion
Hereditary Haemochromatosis (HH) is a genetically inherited disorder [2]. The clinical hallmark of this disease is the gradual accumulation of iron in the vital organs of the body [2]. Mutations in the HFE gene are most frequently associated with HH [2], [17]. In addition, β2M knockout mice also display a iron overload phenotype resembling that observed in HH patients [4]. In this report, we demonstrate that by depleting the cells of β2M by RNAi, HFE localisation is restricted to the ER.
Acknowledgments
We are grateful to Kostas Pantopoulos for the HA-fused HFE vector and Jean Gruenberg for the anti-LBPA antibody. We are also thankful to the members of our laboratory for helpful comments and suggestions. This work was supported by a Health Research Board Grant (RP/2005/107) and a Science foundation Ireland (SFI) Investigator Grant (05/IN.3/B859) to M.M.C.
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