MagA expression attenuates iron export activity in undifferentiated multipotent P19 cells

Abstract

Magnetic resonance imaging (MRI) is a non-invasive imaging modality used in longitudinal cell tracking. Previous studies suggest that MagA, a putative iron transport protein from magnetotactic bacteria, is a useful gene-based magnetic resonance contrast agent. Hemagglutinin-tagged MagA was stably expressed in undifferentiated embryonic mouse teratocarcinoma, multipotent P19 cells to provide a suitable model for tracking these cells during differentiation. Western blot and immunocytochemistry confirmed the expression and membrane localization of MagA in P19 cells. Surprisingly, elemental iron analysis using inductively-coupled plasma mass spectrometry revealed significant iron uptake in both parental and MagA-expressing P19 cells, cultured in the presence of iron-supplemented medium. Withdrawal of this extracellular iron supplement revealed unexpected iron export activity in P19 cells, which MagA expression attenuated. The influence of iron supplementation on parental and MagA-expressing cells was not reflected by longitudinal relaxation rates. Measurement of transverse relaxation rates ( R 2 * and R 2 ) reflected changes in total cellular iron content but did not clearly distinguish MagA-expressing cells from the parental cell type, despite significant differences in the uptake and retention of total cellular iron. Unlike other cell types, the reversible component R 2 ′ ( R 2 * ‒ R 2 ) provided only a moderately strong correlation to amount of cellular iron, normalized to amount of protein. This is the first report to characterize MagA expression in a previously unrecognized iron exporting cell type. The interplay between contrast gene expression and systemic iron metabolism substantiates the potential for diverting cellular iron toward the formation of a novel iron compartment, however rudimentary when using a single magnetotactic bacterial gene expression system like m agA . Since relatively few mammalian cells export iron, the P19 cell line provides a tractable model of ferroportin activity, suitable for magnetic resonance analysis of key iron-handling activities and their influence on gene-based MRI contrast.

Hepcidin-mediated Iron Regulation in P19 Cells is Detectable by Magnetic Resonance Imaging

“…As shown in Fig. 5a, R 2 * increased after iron supplementation compared to untreated cultures (−Fe vs +Fe, p < 0.001), consistent with the avid iron import activity in P19 cells reported in a previous study 15 . Upon removal of extracellular iron supplementation, R 2 * decreased to baseline levels within 4 hours (+Fe vs 4h-Fe, p < 0.001), consistent with reported iron export activity in P19 www.nature.com/scientificreports www.nature.com/scientificreports/ cells 15 and clarifying the time course of iron export.…”

“…Iron particles shorten T 1 and T 2 , resulting in a signal increase in T 1 -weighted images (positive contrast) while producing a signal loss in T 2 -weighted images (negative contrast). While Liu et al 15 showed no influence of changes in extracellular iron on T 1 -weighted images in P19 cell phantoms, if hepcidin activity alters the LIP as proposed above, that might also influence T 1 since it depends on the energy transfer between spins and the surrounding lattice. Thus, if chemical form of surrounding atoms or their compartmentalization is altered under different conditions of iron supplementation and hepcidin-mediated regulation of iron export, this might influence the efficiency of energy transfer and therefore, T 1 .…”

“…Region of interest (ROI) and relaxation rate measurements. Analysis software was previously developed in Matlab 7.9.0 (R2010b) 15,24 and used to determine ROI and to measure R 2 * and R 2 . ROI was outlined to include as many voxels as possible within the sample wells while avoiding the wall of the wells.…”

“…As such, the way cells handle iron in any tissue is expected to affect the MR signal. Having established that P19 cells may be a suitable model for the study of macrophage-like iron homeostasis 15 , we investigated the effect of iron supplementation (representing hemorrhage) and hepcidin (representing pro-inflammatory signaling) on MR transverse relaxation rates 15 .…”

In Vivo Biosynthesis of Inorganic Nanomaterials Using Eukaryotes—A Review

“…al. [ 171 ] extended the efficacy of MTB magA gene when transfected into the genome of mouse multipotent P19 cells to promote the intracellular production of magnetite NPs as an efficient MRI contrast agent.…”