Apo‐lactoferrin stimulates proliferation of mouse crypt cells by activating the MAPK signaling pathway

Abstract

Lactoferrin (Lf), an iron‐binding protein in human milk, plays an important role in intestinal epithelial cell proliferation, but the underlying molecular mechanisms remain unknown. We have shown that the Lf receptor (LfR) mediates Lf endocytosis by Caco‐2 cells via a clathrin‐mediated pathway. The Ras‐dependent extracellular signal‐regulated mitogen‐activated protein kinase (MAPK) cascade is an important pathway transmitting extracellular signals that stimulate cell proliferation. We hypothesized that Lf stimulates proliferation of intestinal epithelial cells by activating Ras‐MAPK and that LfR is involved. Mouse crypt cells were isolated from d 10 pups. Confocal microscopy showed that LfR is expressed and localized at the plasma membrane of crypt cells, specifically binding iron‐free Lf (apo‐Lf) and iron‐saturated Lf (holo‐Lf). BrdU assay showed that Lf stimulates cell proliferation and that apo‐Lf has stronger effect than holo‐Lf. Both LfR antibody blocking and MEK inhibitor (U0126) treatment inhibited stimulation of crypt cell proliferation by apo‐Lf but not holo‐Lf. Western blot showed that apo‐Lf but not holo‐Lf initiates MAPK signaling by ERK‐phosphorylation. Further, both MEK inhibitor and LfR antibody blocking inhibited activation of MAPK signaling by apo‐Lf. Thus, apo‐Lf but not holo‐Lf stimulates intestinal proliferation via initiating MAPK signaling and LfR mediates this process.