Lactoferrin receptor mediates apo- but not holo-lactoferrin internalization via clathrin-mediated endocytosis in trophoblasts.

Lactoferrin receptor (LfR) is expressed in most mammalian tissues including placental trophoblasts and is presumed to mediate the internalization of lactoferrin (Lf). However, the physiological significance of trophoblast LfR is not understood. Using a cytotrophoblast cell model (BeWo) we demonstrated that transfection with LfR siRNA significantly decreased apo- but not holo-Lf uptake compared to mock-transfected controls and that apo- but not holo-Lf significantly increased matrix metalloproteinase (MMP)-2 activity. As Lf functionality is related to the presence (holo-Lf) or absence (apo-Lf) of iron within the Lf molecule, our results suggest that apo-Lf may play a role in cellular invasion. Moreover, we detected LfR (~105 kDa) in association with the plasma membrane and ligand blotting confirmed that Lf binds to a LfR of ~105 kDa. Apo-Lf treatment significantly increased LfR abundance at the plasma membrane and internalization likely occurred via clathrin-mediated endocytosis through early and recycling endosomes as LfR was co-localized with EEA1 and TfR using confocal microscopy and hypertonic medium (0.4 M sucrose) significantly inhibited apo-Lf internalization. In summary, our data demonstrate that apo- but not holo-Lf is internalized by LfR and suggest that, following internalization via LfR, apo-Lf plays a role in cytotrophoblast invasiveness by inducing MMP-2 activity. Moreover, LfR facilitates apo-Lf uptake specifically through clathrin-mediated endocytosis into early endosomes and potentially into a recycling pathway. Taken together, our data provide a new dimension in understanding ligand-dependant function that may be directly related to the ability of LfR to selectively internalize apo- but not holo-Lf.