Iron status in mice carrying a targeted disruption of lactoferrin.

Lactoferrin is an 80-kDa member of the transferrin family of nonheme iron-binding glycoproteins (34). The most extensively characterized member of this family is transferrin, which functions as the major iron-binding and transport protein in serum and which is essential for iron delivery to erythrocytes and developing neuronal cells via the transferrin-transferrin receptor cycle (7, 28, 45). Despite sharing a high degree of homology at the amino acid sequence level (34) and the three-dimensional conformational level (1, 2, 4), lactoferrin differs from transferrin in many aspects, including localization and functional activity. Lactoferrin expression is first detected during murine embryonic development in the preimplantation embryo and is expressed during the latter half of gestation in the developing hematopoietic, digestive, and respiratory systems (47). In adults of many species, lactoferrin is expressed and secreted by glandular epithelial cells (27, 32). Lactoferrin is also expressed during late-stage granulopoiesis and is stored in the secondary granules of mature neutrophils, from which it is released into the circulation upon neutrophil degranulation (31). While many diverse physiological functions have been proposed for lactoferrin, including the regulation of intestinal iron uptake, antimicrobial activity, regulation of cellular growth and differentiation, anti-inflammatory and immunomodulatory activities, and protection against cancer (reviewed in references 6, 8, 23, 27, 48, and 49), the precise mechanism of action of lactoferrin in many of these processes remains poorly understood. In particular, a long-standing controversy exists as to the role of lactoferrin in intestinal iron homeostasis. Iron is an essential element required for many metabolic functions, yet it can be toxic in excess, promoting free radical formation. Clearly, tight regulation of iron homeostasis is essential for protecting against free radical-induced cellular damage while maintaining proper cellular function. In higher organisms, iron homeostasis is regulated at the level of absorption from the diet in the proximal small intestine in response to body iron status (50). Recently, many of the genes involved in intestinal iron uptake were identified, and loss-of-function mutations of these genes in rodents have greatly enhanced the understanding of iron homeostasis at the molecular level (3). In particular, a divalent metal iron transporter (DMT-1) has been identified as a major protein involved in intestinal nonheme iron uptake at the apical surface, and loss-of-function mutations in DMT-1 have been found to result in severe iron-deficiency anemia, due in part to a primary defect in intestinal iron uptake (3, 10, 18, 19, 22). Intestinal iron homeostasis is also regulated by the product of the hemachromatosis gene, HFE, a novel major histocompatibility complex class 1-like protein that is mutated in the majority of patients with hereditary hemachromatosis (17). Mice with a targeted mutation in the HFE gene exhibit classic symptoms of hemachromatosis, including excessive intestinal iron uptake resulting in increases in serum iron levels, transferrin saturation, and liver iron stores (52). The molecular control of iron homeostasis by HFE is incompletely understood, but it has been proposed that the association of HFE and the transferrin receptor on the basolateral surface of duodenal crypt cells leads to sensing of body iron requirements, resulting in a feedback alteration in the iron absorption capacity of mature enterocytes (21, 38). The strong iron-binding properties of lactoferrin, together with the high iron bioavailability and abundant concentration of lactoferrin in breast milk, have prompted speculation that this protein may also be involved in the delivery of iron to the neonate (23, 27). Further, it has been observed that lactoferrin is relatively resistant to proteolysis in the gastrointestinal tract (16) and that lactoferrin, but not transferrin, can deliver bound iron to human intestinal epithelial cells (14). In addition, a specific receptor for lactoferrin has been identified on the apical surface of enterocytes from many species (23), and the cloning of a human enterocyte receptor was recently reported (42). In vitro experiments demonstrating the increased uptake of iron-saturated lactoferrin in Caco-2 cells transfected with this receptor further support the view that lactoferrin is involved in intestinal iron uptake (42). However, it has also been proposed that the iron-binding properties and stability of lactoferrin are responsible for iron sequestration rather than delivery in the gastrointestinal tract, thus preventing excessive iron uptake (9). Considerable debate has persisted because of a lack of a relevant model for adequately assessing the essential physiological role of this protein in intestinal iron homeostasis. To directly assess the functions of lactoferrin in vivo, we have generated a mouse model of lactoferrin deficiency by using gene targeting techniques. In the study described here, we show that lactoferrin is not required for intestinal iron uptake and that the iron status in the absence of lactoferrin is relatively normal, results which are inconsistent with a major role of this protein in iron homeostasis.