Iron deficiency and iron overload are prevalent conditions around the world with various adverse consequences. The need of iron as a critical nutrient and the necessity for iron homeostatic regulation is well appreciated. Iron homeostasis is solely regulated through intestinal iron absorption through incompletely understood mechanisms. Ferroportin 1 (FPT1) is the iron transporter exporting iron across the basal-lateral membrane of enterocytes to the systemic circulation. Mutations of human FPT1 are associated with type 4 hereditary hemochromatosis suggesting that FPT1 plays a specific role in iron homeostatic regulation. The long-term goal of this proposal is to elucidate the mechanism by which the function and the expression of FPT1 are regulated.
The specific aims are to determine the effect of iron and hepcidin on the expression and subcellular localization of FPT1 in relation to intestinal iron absorption. Iron and/or Hepcidin induced changes in the expression, migration and function of FPT1 will be determined in rat intestine and in Caco2 cells including Caco2 cells transfected with vectors expressing wild-type or mutant FPT1 and its interacting proteins. Confocal microscopy and velocity sedimentation will be used to analyze differences in the migration of wild-type and mutant FPT1. Changes in intestinal iron absorption and iron transport across Caco2 cells will be monitored in relation to subcellular FPT1 sites. Hepcidin suppression of FPT1 expression and function will be examined to determine the mechanism of FPT1 regulation by hepcidin. Finally, the interactions among FPT1, liver fatty acid biding protein, heme oxygenase and hephaestin will be verified by confocal immunofluorescent microscopy, co-immunoprecipitation, and mammalian two hybrid assay and the function of these protein-protein interactions in intestinal iron absorption will be determined by iron transport across the Caco2 cell layer. The results should reveal mechanisms involved in the regulation and function of FPT1 and extend our understanding of intestinal iron absorption and body iron homeostatic regulation. ? ?
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