Progress 01/01/05 to 11/30/09
Outputs
OUTPUTS: To characterize the functions of Heph and Fpn in the process of intestinal iron absorption, we created cells stably expressing Heph and Fpn using tetracycline inducible system. When Heph-transfected cells were treated with tetracyline, the levels of Heph protein were increased and Heph's ferroxidase was significantly increased with copper treatment. Similarly, when Fpn-transfected Caco-2 cells were treated with tetracycline, Fpn protein expression was increased without changing the levels of proteins involved in iron transport. To determine interactions of Fpn and Heph in intestinal cells, we initially generated cells lacking Heph using tetracycline inducible system containing H1 promoter. When Heph siRNA-transfected cells were treated with tetracycline, the levels of Heph protein were significantly decreased but subcellular distribution of Fpn protein was not changed by the decrease of Heph protein level. Fpn protein was primarily located on the basolateral membrane of enterocytes in these Heph deficient cells. Similarly, effects of Fpn on the localization of Heph were studied in Fpn deficient cells. Fpn deficient cells were generated using a tetracycline-inducible system. When Fpn siRNA-transfected cells were treated with tetracycline, Fpn protein levels were significantly decreased. The subcellular distribution of Heph protein was not significantly altered in these Fpn deficient cells. To examine whether Heph is physically associated with Fpn, we initially performed co-immunoprecipitation experiments in cells expressing the GFP-tagged Fpn. Our Western-blot analysis of total cell extracts showed that the endogenous Heph was present in both non transfected control cells and transfected cells expressing GFP-Fpn, whereas immunoprecipitation of cell proteins with the anti-GFP antibody pulled down most of the GFP-Fpn molecules and precipitated Heph protein. The immunoprecipitation of non-transfected cells with the anti-GFP antibody did not yield any immunoreactive bands for Heph. To further investigate interactions between Fpn and Heph, we performed experiments using fluorescence energy transfer (FRET). We prepared appropriate fluorescent probes by attaching CFP (cyan fluorescent protein) to the C-terminus of Heph and YFP (yellow fluorescent protein) to the C-terminus of Fpn. In cells stably expressing CFP-Heph, we first assessed CFP fluorescence, pixel by pixel (using Fluoview 1000 confocal laser scanning microscope, Olympus). In cells stably expressing both CFP-Heph and YFP-Fpn, we then reassessed fluorescence of each pixel at the CFP donor emission wavelength. A YFP-induced decrease in CFP emission would suggest that the two proteins are associated. We further investigated whether association of these proteins are required for the iron transport. We deleted 194 amino acid residues of Heph as seen in sex linked anemia mice and determined its effects on its interactions with Fpn and iron transport. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Our study showed that both Fpn and Heph are crucial molecules for intestinal iron absorption. We determined functions of Heph and Fpn in the process of intestinal iron absorption by creating cells overexpressing Heph and Fpn using tetracycline inducible system. When Heph-transfected cells were treated with tetracyline, the levels of Heph protein were increased and Heph's ferroxidase was significantly increased with copper treatment. These results suggest that Heph has a ferroxidase function. The transport of iron across the basolateral membrane was significantly increased in Heph overexpressing cells pretreated with copper. Similarly, when Fpn-transfected Caco-2 cells were treated with tetracycline, Fpn protein expression was increased and transepithelial iron transport was induced without changing the levels of proteins involved in iron transport. To determine interactions of Fpn and Heph in intestinal cells, we generated cells lacking either Heph or Fpn using tetracycline inducible system. Although Heph protein was significantly decreased, the subcellular distribution of Fpn protein was not changed. Fpn protein was primarily located on the basolateral membrane of enterocytes in these Heph deficient cells. Iron transport was reduced when Heph's ferroxidase was decreased. The levels of DMT1 and TfR1 protein were not changed in Heph-deficient cells. Similarly, Heph protein was not significantly altered in Fpn deficient cells. The transepithelial iron transport was significantly decreased in Fpn deficient cells. Together, these results suggest that levels of Fpn protein are important factor affecting iron transport across the enterocyte. However, these results indicate that Fpn (Heph) does not requre Heph (Fpn) for it's biosynthesis and basolateral localization. Our data suggest cells overexpressing / lacking Fpn and Heph that were created using tetracycline inducible system are useful models to study the mechanism of intestinal iron absorption. We also examined whether Heph is physically associated with Fpn by performing co-immunoprecipitation experiments using cells expressing the GFP-tagged Fpn. Our co-immunoprecipitation results suggested the possibility that Fpn might interact with Heph in the process of iron exit across the basolateral membrane of intestinal absorptive cell. To further investigate interactions between Fpn and Heph, we performed experiments using fluorescence energy transfer (FRET). Our FRET experiments showed that two proteins, Fpn and Heph, physically interact on the basolateral membrane of intestinal enterocyte. Since Heph protein is associated with Fpn protein, we next investigated whether association of these proteins are required for the iron transport. We deleted 194 amino acid residues of Heph as seen in sex linked anemia mice and determined its effects on interactions with Fpn and iron transport. We found that 194 amino acid truncated Heph did not interact with Fpn and was not able to stimulate iron transport suggesting that interactions of Fpn with Heph might be a crucial factor in the process of iron transport.
Publications
- Beard J. and Han O. Systemic iron status. (2009) Biochim Biophys Acta: 1790(7):584-8
- Bergmann OM, Mathahs MM, Broadhurst KA, Weydert JA, Wilkinson N, Howe JR, Han O, Schmidt WN, Brown KE. Altered expression of iron regulatory genes in cirrhotic human livers: clues to the cause of hemosiderosis(2008) Lab Invest;88(12):1349-57.
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Progress 01/01/06 to 12/31/06
Outputs
We generated cell lines expressing tetracycline inducible ferroportin-1 (FPN-1) an iron exporter or hephaestin (Heph) a ceruloplasmin homologue ferroxidase. Our data showed that the exogenous proteins function as endogenous ones. For example, ferroxidase activity was significantly increased in cells overexpressing Heph protein compared to the control. We also created cells lacking FPN-1 or Heph using tetracycline inducible system. We are currently investigating whether the levels of these proteins are the determining factors for iron export across the basolateral membrane of the enterocyte. We previously showed that FPN-1 and Heph colocalized with transferrin receptor-1 (TfR-1) on the basolateral membrane of the human enterocyte. Our co-immunoprecipitation studies also showed the possibility that FPN-1 and Heph associate with TfR-1 on the basolateral membrane of enterocytes. However, our fluorescence resonance energy transfer (FRET) studies revealed that neither of
FPN-1 nor Heph interacts with TfR-1 in intestinal cells. Our previous results demonstrated that Heph colocalizes with GFP-tagged FPN-1 on the basolateral membrane of intestinal cells, which indicate the possibility that these two proteins interact in the process of iron absorption across the enterocyte. To assess if FPN-1 and Heph interact each other in the process of iron absorption, we created FPN-1 tagged with yellow fluorescent protein (YFP) and Heph tagged with cyan fluorescent protein (CFP). We are currently selecting transfected cell lines with FPN-1-YFP and / or Heph-CFP to determine their interactions in the process of iron export using FRET.
Impacts
Our data suggest that cells overexpressing / lacking FPN-1 and Heph that were created using tetracycline inducible system are useful models to study the mechanism of iron absorption. Our FRET studies demonstrate that although FPN-1 and Heph are colocalized with TfR-1 but they are not directly interact with TfR-1. By understanding the mechanisms by which iron export across the basolateral membrane of enterocyte, we will be able to develop more effective strategies to promote nutritional status and iron absorption during life cycle as well as during various patho-physiological conditions.
Publications
- Eun-Young Kim, Okhee Han (2008) Effects of iron chelators on iron export across the basolateral membrane of human intestinal Caco-2 cells. FASEB J, Experimental Biology 2008, San Diego, CA. April, 2008
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Progress 01/01/05 to 01/01/06
Outputs
We localized an iron exporter ferroportin-1 (FPN-1) and a multi-copper oxidase hephaestin (Heph) in the intestinal absorptive cells using affinitive purified FPN-1 and Heph antibodies. Our confocal microscope study showed that FPN-1 and Heph are located on the basolateral membrane, and they are colocalized and associated with transferrin receptor (TfR) in human intestinal absorptive cell like fully differentiated Caco-2 cells grown on microporous membrane inserts. To determine whether FPN-1 is colocalized with Heph in intestinal absorptive cells, we stably transfected Caco-2 cells with human FPN-1 modified by the addition of GFP at the C-terminal. We found that GFP-tagged FPN-1 is located on the basolateral membrane and it is associated with Heph in human intestinal absorptive cell like Caco-2 cells. Furthermore, the immuno-coprecipitation study revealed that Heph is coprecipitated with GFP-tagged FPN-1, which suggests the possibility that FPN-1 might interact with
Heph in the process of iron exit across the basolateral membrane of intestinal absorptive cell. We are currently selecting stably transfected cell lines to identify the exact functions of FPN-1 and Heph in the process of intestinal iron absorption.
Impacts
Our data suggest that FPN-1 and Heph might interact in the process of iron exit across the basolateral membrane of intestinal absorptive cells. By understanding cellular and molecular mechanisms of intestinal iron absorption, we will be able to develop more effective strategies to promote nutritional status and iron absorption.
Publications
- Han,O and Kim, EY. 2007. Colocalization of Ferroportin-1 with Hephaestin on the Basolateral Membrane of Human Intestinal Absorptive Cells. Journal of Cellular Biochemistry. (In Press)
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