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

Rulan Jiang, Veronica Lopez, Shannon L. Kelleher, Bo Lönnerdal

Research output: Contribution to journalArticle

78 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)3022-3031
Number of pages10
JournalJournal of Cellular Physiology
Volume226
Issue number11
DOIs
StatePublished - Nov 2011

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Clathrin
Lactoferrin
Caco-2 Cells
Cell proliferation
Endocytosis
Cell Proliferation
Iron
Enterocytes
Small Interfering RNA
lactoferrin receptors
Vesicular Transport Adaptor Proteins
Biotinylation

ASJC Scopus subject areas

  • Clinical Biochemistry
  • Cell Biology
  • Physiology

Cite this

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. / Jiang, Rulan; Lopez, Veronica; Kelleher, Shannon L.; Lönnerdal, Bo.

In: Journal of Cellular Physiology, Vol. 226, No. 11, 11.2011, p. 3022-3031.

Research output: Contribution to journalArticle

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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.",
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