Apo- and Holo-lactoferrin Stimulate Proliferation of Mouse Crypt Cells but Through Different Cellular Signaling Pathways

Lactoferrin (Lf), an iron-binding glycoprotein in human milk, plays an important role in intestinal epithelial cell proliferation, but the underlying molecular mechanisms remain unknown. Mitogen-activated protein kinase (MAPK)/extracellular signal-regulated protein kinases (ERK) and the phosphoinositide 3-kinase (PI3K) cascades are chief pathways controlling cell proliferation. We hypothesized that Lf stimulates proliferation of intestinal epithelial cells by activating ERK and PI3K signaling cascades and that lactoferrin receptor (LfR) is involved in these processes. Confocal microscopy showed that LfR is expressed and localized at the plasma membrane of mouse crypt cells isolated from 7 to 10 days old pups, specifically binding both iron-free Lf (apo-Lf) and iron-saturated Lf (holo-Lf). Using the BrdU assay, we observed that apo-Lf had a stronger stimulatory effect than holo-Lf on proliferation of crypt cells. Both function-blocking antibody against LfR and MEK inhibitor (U0126) resulted in inhibitory effects on apo-Lf-induced cell proliferation, whereas PI3K inhibitor (LY294002) significantly decreased both apo- and holo-Lf-induced proliferation. Although both apo- and holo-Lf up-regulated transcription of cyclin D1, an effector of ERK1/2 and PI3K/Akt signaling cascades, only apo-Lf initiated ERK1/2 signaling and both apo- and holo-Lf were capable of activating the PI3K/Akt signaling pathway. Function-blocking antibody against LfR and LfR siRNA inhibited apo-Lf induced activation of the ERK1/2 signaling pathway, suggesting that LfR is involved in apo-Lf induced activation of the ERK1/2, but not the PI3K signaling pathway. In conclusion, apo-Lf and holo-Lf stimulate intestinal proliferation but through different signaling pathways.