In mammals, effective acquisition and handling of iron is critical for survival.Consequently, iron handling proteins are exquisitely responsive to changes iniron concentrations. Regulation occurs at both the transcriptional andtranslational levels. The work described in this thesis focuses on two ironhandling proteins that are central to iron regulation and homeostasis, namelydivalent metal transporter 1 (DMT1) and transferrin (Tf). Experiments wereperformed independently to test two aims and accordingly the outcomes ofthese experiments are described separately.1. To determine the cellular location of heterologously expressed ironinduced(DMT1) splice variants.Two novel DMT1 splice variants, lacking exon 5 (DMT1Delta5) and exons 12(DMT1Delta12) were induced by treating cells with desferrioxamine (DFO), atreatment that lowers the cellular iron concentration. Previously, DMT1Delta5 andDMT1Delta12 have been described in patients with DMT1 mutations, however,these studies did not establish a direct link between mutations, anaemia orsplicing. Therefore, as a first step towards understanding their function weaimed to engineer DMT1Delta5 and DMT1Delta12 constructs tagged with mRFP andexpress them in non-polarised and polarised cell models.Heterologous expression of DMT1 in WKPT cells localised predominantly tostructures resembling the late endosomes and lysosomes as previously shown.In contrast, mRFP-DMT1Delta5 localised to the nuclei and led to cell death after 16hours post transfection in WKPT cells. However, no expression of mRFPDMT1Delta12was detected in hamster embryonic kidney 293 or WKPT cells.These studies suggest that low-iron induced splicing events may differentiallyaffect subcellular localisation of DMT1 in polarised and non-polarised cells.2. To determine the mechanism of transferrin uptake into the WKPT renalepithelial cell line.Multi-ligand receptors, megalin/cubilin are suggested to be the mediators of Tfuptake in the proximal tubule. However in mice, the TfR1 has been shown tolocalise at the apical membrane of the proximal tubule epithelia. Therefore, weaimed to determine the contribution of megalin/cubilin to cellular Tf delivery inthe PT. Experiments were performed in vitro, using a cell line (WKPT-0293Cl.2) derived from the proximal tubule of rats. Ligands of megalin/cubilin,receptor associated protein (RAP) and Tf were used for uptake studies.Uptake studies confirmed that when iron is replete, megalin/cubilin are themain mode of Tf (Tfmeg/cub) uptake in PT cells. However, when cellular iron wasreduced, a switch to a RAP insensitive receptor (RIR) upregulated Tf (TfRIR)uptake, leading to an increase in intracellular Tf. Further experiments showedthat Tf enters a common pathway irrespective of the receptor in which itsuptake is mediated. However, TfRIR was retained for longer in comparison toTfmeg/cub, highlighting a key difference between the two intracellular pathways ofTfmeg/cub and TfRIR. In summary, data suggested that megalin/cubilin are thedominant means by which Tf is endocytosed when iron was replete. Howeverwhen cellular iron is restricted, TfR1 switches to become the principal receptor for Tf endocytosis.
|Date of Award||31 Dec 2013|
- The University of Manchester
|Supervisor||Craig Smith (Supervisor) & Nicholas Ashton (Supervisor)|
The characterisation of the novel effects of low-iron on renal epithelial cells
Haley, M. (Author). 31 Dec 2013
Student thesis: Phd