title: Apo- and holo-lactoferrin are both internalized by lactoferrin receptor via clathrin-mediated endocytosis but differentially affect ERK-signaling and cell proliferation in Caco-2 cells.
abstract: Lactoferrin (Lf) is a major iron-binding and multi-functional protein in exocrine fluids such as breast milk and mucosal secretions. The functions of Lf appear dependent upon the iron saturation of the Lf protein and are postulated to be mediated through Lf internalization by a Lf receptor (LfR). However, mechanisms by which LfR mediates Lf internalization in enterocytes are unknown. We now demonstrate that a LfR previously cloned from the small intestine mediates Lf endocytosis in a human enterocyte model (Caco-2 cells). LfR was detected at the plasma membrane by cell surface biotinylation; both apo-Lf and holo-Lf uptake were significantly inhibited in cells transfected with LfR siRNA. Treatments of hypertonic sucrose and clathrin siRNA and co-immunoprecipitation of LfR with clathrin adaptor AP2 indicate that LfR regulates Lf endocytosis via clathrin-mediated endocytosis. Although both iron-free Lf (apo-Lf) and iron-saturated Lf (holo-Lf) enter Caco-2 cells via a similar mechanism and no significant differences were observed in the binding and uptake of apo- and holo-Lf in Caco-2 cells, apo-Lf but not holo-Lf stimulates proliferation of Caco-2 cells. Interestingly, apo-Lf stimulated extracellular signal-regulated mitogen-activated protein kinase (ERK) cascade to a significantly greater extent than holo-Lf and the apo-Lf induced proliferation was significantly inhibited by an ERK cascade inhibitor (U0126) and clathrin siRNA. Taken together, our data suggest that LfR is a major pathway through which Lf is taken up by enterocytes, which occurs independently of iron saturation through clathrin-mediated endocytosis. The differential effects of apo- and holo-Lf are not due to differences in cellular internalization mechanisms.
authors.affiliation: Department of Nutrition, University of California, Davis, California 95616, USA.
authors.forename: Rulan
authors.initials: R
authors.lastname: Jiang
authors.forename: Veronica
authors.initials: V
authors.lastname: Lopez
authors.forename: Shannon L
authors.initials: SL
authors.lastname: Kelleher
authors.forename: Bo
authors.initials: B
authors.lastname: Lönnerdal
mesh_terms: D001059:Apoproteins / Q000378:metabolism*; D002070:Butadienes / Q000494:pharmacology; D018938:Caco-2 Cells; D049109:Cell Proliferation / Q000187:drug effects; D002966:Clathrin / Q000378:metabolism*; D004705:Endocytosis / Q000187:drug effects*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism*; D006801:Humans; D007501:Iron / Q000378:metabolism; D007781:Lactoferrin / Q000378:metabolism*; D009570:Nitriles / Q000494:pharmacology; D034741:RNA, Small Interfering / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects
chemical_list: D001059:Apoproteins; D002070:Butadienes; D002966:Clathrin; D004791:Enzyme Inhibitors; D009570:Nitriles; D034741:RNA, Small Interfering; D011956:Receptors, Cell Surface; C113580:U 0126; C039223:apolactoferrin; C492629:hololactoferrin, human; C038340:lactoferrin receptors; D007501:Iron; D048049:Extracellular Signal-Regulated MAP Kinases; D007781:Lactoferrin