Association of HFE protein with transferrin receptor in crypt enterocytes of human duodenum

Abstract

In hereditary hemochromatosis (HH), intestinal absorption of dietary iron is increased, leading to excessive iron accumulation in tissues and resultant organ damage. The HFE protein, which is defective in HH, normally is expressed in crypt enterocytes of the duodenum where it has a unique, predominantly intracellular localization. In placenta, the HFE protein colocalizes with and forms a stable association with the transferrin receptor (TfR), providing a link between the HFE protein and iron transport. In the present study, we examined the relationship of the HFE protein to the TfR in enterocytes of the human duodenum and measured the uptake of transferrin-bound iron and ionic iron by isolated crypt and villus enterocytes. Immunocytochemistry showed that the HFE protein and TfR both are expressed in the crypt enterocytes. Western blots showed that, as was the case in human placenta, the HFE protein in crypt enterocytes is physically associated with the TfR and with beta2-microglobulin. The crypt cell fraction exhibited dramatically higher transferrin-bound iron uptake than villus cells. On the other hand, the villus cells showed 2-3 times higher uptake of ionic iron than crypt cells. We propose that the HFE protein modulates the uptake of transferrin-bound iron from plasma by crypt enterocytes and participates in the mechanism by which the crypt enterocytes sense the level of body iron stores. Impairment of this function caused by HFE gene mutations in HH could provide a paradoxical signal in crypt enterocytes that programs the differentiating enterocytes to absorb more dietary iron when they mature into villus enterocytes.

Figure 1

Immunohistochemistry of HFE protein and TfR in human duodenum. Immunoperoxidase staining of the HFE protein shows that the most intense signal is confined to the crypt enterocytes (A and C). The TfR shows wider distribution along the crypt-villus axis (B), but heavier staining at the basolateral surface of the crypt cells (D). Double immunostaining of the HFE protein and TfR demonstrates that both proteins are expressed in the same crypt enterocytes (E). The HFE protein shows a strong perinuclear staining pattern (C and E), whereas the TfR shows more diffuse intracellular as well as basolateral membrane-associated immunoreactions (small arrows in D). Leukocytes in the cellular lamina propria express both proteins (large arrows in C and D). The green and red colors in E indicate the HFE protein and TfR immunoreactions, respectively. Colocalization of HFE and TfR proteins is indicated by the yellow color. c, crypt region; v, villus. (Bars: A and B = 100 μm; C and D = 20 μm; and E = 25 μm.)

Figure 2

Expression of HFE protein and TfR in crypt and villus cells of human duodenum. Aliquots of cell homogenates from fraction 1 (villus cells) and fraction 4 (crypt cells) containing 20 μg of protein were analyzed by SDS/PAGE under reducing conditions followed by immunostaining using the anti-HFE-CT16 or anti-TfR antibodies (A). Both HFE protein and TfR show more prominent signals in the crypt cells. (B) A confocal laser scanning microscopy image of the HFE protein and TfR in a crypt cell. Blue color (reflection image) demonstrates the location of two cells. The green color for HFE and red color for TfR produce a strong whitish yellow color reaction where the immunostainings colocalize. (Bar = 25 μm.)

Figure 3

Western blot of TfR and β₂M coimmunoprecipitated with HFE from lysates of crypt and villus cell fractions from human duodenum. The TfR and β₂M in immunocomplexes isolated by immunoprecipitation with the anti-human HFE-CT16 antibody were identified on Western blot by a mixture of anti-TfR and anti-human β₂M antibodies.

Figure 4

Uptake of transferrin-bound iron and ionic iron by fractionated mucosal cells. (A) The uptake of transferrin-bound iron is low in the first three fractions (F1–F3) and is much higher in the fraction most greatly enriched for crypt cells (F4). (B) Uptake of ionic iron (⁵⁹Fe-NTA) is higher in the fractions having the greatest enrichment for villus cells (F1–F3).