Mary Yeh

Iron feeding induces ferroportin 1 and hephaestin migration and interaction in rat duodenal epithelium

Intestinal iron absorption involves proteins located in the brush border membrane (BBM), cytoplasm, and basolateral membrane (BLM) of duodenal enterocytes. Ferroportin 1 (FPN1) and hephaestin (Heph) are necessary for transport of iron out of enterocytes, but it is not known whether these two proteins interact during iron absorption. We first examined colocalization of the proteins by cotransfection of HEK293 cells with pDsRed-FPN1 with pEmGFP-Heph or with the COOH-terminal truncated pEmGFP-HephDelta43 or -HephDelta685 and found that FPN1 and Heph with or without the COOH terminus colocalized. In rat duodenal enterocytes, within 1 h of iron feeding prominent migration of FPN1 from the apical subterminal zone to the basal subnuclear zone of the BLM occurred and increased to at least 4 h after feeding. Heph exhibited a similar though less prominent migration after iron ingestion. Analysis using rat duodenal epithelial cell sheets demonstrated that 1) by velocity sedimentation ultracentrifugation, FPN1 and Heph occupied vesicles of different sizes prior to iron feeding and migrated to similar fractions 1 h after iron feeding; 2) by blue native/SDS-PAGE, FPN1, and Heph interacted to form two complexes, one containing dimeric FPN1 and intact Heph and the other consisting of monomeric FPN1 and a Heph fragment; and 3) by immunoprecipitation, anti-Heph or anti-FPN1 antiserum coimmunoprecipitated FPN1 and Heph. Thus the data indicate that FPN1 and Heph migrate and interact during iron feeding and suggest that dimeric FPN1 is associated with intact Heph.

DMT1 (IRE) expression in intestinal and erythroid cells is regulated by peripheral benzodiazepine receptor-associated protein 7.

The divalent metal transporter 1 (DMT1) is essential for cellular uptake of iron, mediating iron absorption across the duodenal brush border membrane. We have previously shown that with iron feeding DMT1 in the brush border membrane undergoes endocytosis into the subapical compartment of enterocytes. To understand the mechanisms of iron-induced endocytosis of DMT1, we used the yeast two-hybrid system to find proteins that interact with DMT1 and isolated from a rat duodenal cDNA library a protein that interacts specifically with the IRE containing isoform of DMT1 {DMT1 [iron-responsive element (IRE)]}. The protein (Genbank AY336075) is 97.5% identical with peripheral benzodiazepine receptor-associated protein 7 (PAP7), a protein that interacts with the peripheral benzodiazepine receptor. PAP7 is ubiquitously expressed in the rat and in multiple cell lines with consensus sequences including a nuclear localization signal and a Golgi dynamic domain. PAP7, expressed on the brush border of rat duodenum, copurified with DMT1 in brush border membrane vesicles, and following iron feeding, was internalized in parallel with the internalization of DMT1. To determine if PAP7 plays a role in cellular iron metabolism, we downregulated PAP7 expression in K562 cells with small interfering RNA. Following the decrease in PAP7 protein, DMT1 (IRE) protein but not mRNA was significantly downregulated but without effect on DMT1 (non-IRE), transferin (Tf)R1, or ferritin expression. Lowered levels of PAP7 resulted also in decreased cell proliferation and G(1) cell cycle arrest. These data are consistent with PAP7 interacting with DMT1 (IRE) and regulating DMT1 (IRE) expression in K562 cells by modulating expression of DMT1 (IRE) protein.

Hypoxia-inducible factor-2α and iron absorptive gene expression in Belgrade rat intestine

The divalent metal transporter (DMT1, Slc11a2) is an important molecule for intestinal iron absorption. In the Belgrade (b/b) rat, the DMT1 G185R mutation markedly decreases intestinal iron absorption. We used b/b rats as a model to examine the genes that could be compensatory for decreased iron absorption. When tissue hypoxia was assayed by detecting pimonidazole HCl adducts, the b/b liver and intestine exhibited more adducts than the +/+ rats, suggesting that hypoxia might signal altered gene expression. Total RNA in the crypt-villus bottom (C-pole) and villus top (V-pole) of +/+, b/b, and iron-fed b/b rats was isolated for gene array analyses. In addition, hepatic hepcidin and intestinal hypoxia-inducible factor-α (Hifα) expression were examined. The results showed that expression of hepatic hepcidin was significantly decreased and intestinal Hif2α was significantly increased in b/b and iron-fed b/b than +/+ rats. In b/b rats, the expression of Tfrc mRNA in the C-pole and of DMT1, Dcytb, FPN1, Heph, Hmox1, and ZIP14 mRNAs in the V-pole were markedly enhanced with increases occurring even in the C-pole. After iron feeding, the increased expression found in b/b rats persisted, except for Heph and ZIP14, which returned to normal levels. Thus in b/b rats depressed liver hepcidin production and activated intestinal Hif2α starting at the C-pole resulted in increasing expression of iron transport genes, including DMT1 G185R, in an attempt to compensate for the anemia in Belgrade rats.

Expression of intestinal brush-border membrane hydrolases and ferritin after segmental ischemia-reperfusion in rats

Jejunal expression of three brush-border membrane (BBM) enzymes, intestinal alkaline phosphatase (IAP), lactose-phlorizin hydrolase (LPH), and sucrase-isomaltase (SI), and a cytosolic protein, ferritin (Ft), was investigated after transient segmental ischemia-reperfusion (I/R). I/R reduced mucosal IAP, LPH, and SI mRNAs to 36%, 11%, and 38% of normal jejunal levels after 3 h of reperfusion and to 22%, 8%, and 51% of normal jejunal levels after 6 h of reperfusion, respectively. Intriguingly, in the internal control jejunum IAP and LPH mRNAs also decreased significantly. LPH and SI mRNA rapidly recovered to levels significantly higher than those of normal jejunum at 12 h, whereas IAP mRNA levels did not recover until 48 h. Enzyme activity paralleled changes in mRNA levels in the ischemic reperfused jejunum. Electrophoretic mobility shift assays showed that I/R significantly increased SI footprinting 1 (SIF1) binding activity. The mobility of one of the DNA-protein complexes was further retarded in the presence of anti-Cdx-2 antibody, suggesting that either Cdx-2 or a related protein was interacting with the SIF1 sequences. Similar to BBM enzymes, cytosolic Ft mRNA and protein were significantly decreased at 3 and 6 h after I/R. By 12 h, Ft mRNA, but not Ft protein, had increased to higher than normal levels. We conclude that a rapid recovery of BBM mRNAs and enzymes occurs in regenerating mucosa after upper villus damage. The increase of SIF1 binding protein activity after I/R may enhance SI, and perhaps LPH, gene transcription. The expression of Ft is regulated at both pretranslational and translational levels.Jejunal expression of three brush-border membrane (BBM) enzymes, intestinal alkaline phosphatase (IAP), lactose-phlorizin hydrolase (LPH), and sucrase-isomaltase (SI), and a cytosolic protein, ferritin (Ft), was investigated after transient segmental ischemia-reperfusion (I/R). I/R reduced mucosal IAP, LPH, and SI mRNAs to 36%, 11%, and 38% of normal jejunal levels after 3 h of reperfusion and to 22%, 8%, and 51% of normal jejunal levels after 6 h of reperfusion, respectively. Intriguingly, in the internal control jejunum IAP and LPH mRNAs also decreased significantly. LPH and SI mRNA rapidly recovered to levels significantly higher than those of normal jejunum at 12 h, whereas IAP mRNA levels did not recover until 48 h. Enzyme activity paralleled changes in mRNA levels in the ischemic reperfused jejunum. Electrophoretic mobility shift assays showed that I/R significantly increased SI footprinting 1 (SIF1) binding activity. The mobility of one of the DNA-protein complexes was further retarded in the presence of anti-Cdx-2 antibody, suggesting that either Cdx-2 or a related protein was interacting with the SIF1 sequences. Similar to BBM enzymes, cytosolic Ft mRNA and protein were significantly decreased at 3 and 6 h after I/R. By 12 h, Ft mRNA, but not Ft protein, had increased to higher than normal levels. We conclude that a rapid recovery of BBM mRNAs and enzymes occurs in regenerating mucosa after upper villus damage. The increase of SIF1 binding protein activity after I/R may enhance SI, and perhaps LPH, gene transcription. The expression of Ft is regulated at both pretranslational and translational levels.