Apolactoferrin in Mother’s Milk Induces HIF Signaling in Neonate Animals

Introduction: Lactoferrin (LF) is the cationic transferrin of exocrine secretions (milk, lacrimal fluid, saliva, etc.) and neutrophilic leukocytes. In breast milk and neutrophilic granules, apo-LF (iron-free form) makes up 90% of the whole bulk of LF.¹ The extreme affinity of human apo-LF toward iron ions (K_a ~ 10²² M⁻¹) is usually considered in the light of the protein’s bacteriostatic effect.

Iron-chelating features of LF are often compared with the properties of a bacterial siderophore desferrioxamine (DFO).² Since 1993, DFO is regarded as a pharmacological mimetic of hypoxia, since hypoxia-inducible factor-1 α (HIF-1a) becomes stabilized in its presence³ At normoxia, HIF-1a is hydroxylized by iron-sensitive hydroxylases termed PHD and FIH, after which it undergoes ubiquitination and proteasomal degradation. Under hypoxia or iron deficiency, HIF-1a is not modified by PHD or FIH, but enters the nucleus and binds to the constitutive protein HIF-1b to stimulate expression of ~200 genes, half of which secure the survival of a cell under stress. Among those are genes conditioning iron metabolism and erythropoiesis (TF and its receptor, ferritin, ceruloplasmin, erythropoietin [EPO], heme oxygenase), glucose metabolism, angiogenesis, vasodilatation, cell growth, etc. (Lee et al, 2004).⁴

A recent work of our group shows that human apo-LF plays a role of physiological mimetic of hypoxia by stabilizing HIF-1a and HIF-2a, which results in expression of target genes, coding for CP and EPO.^5,6

Here, we present data concerning the effect of foreign apo-LF contained in the milk of lactating rats on neonate pups.

Materials and Methods: Human LF was purified as described by Zakharova et al.⁶ Bovine LF was purified in a similar manner. Native LF was mostly represented by its apo-form (ca. 75% iron-free protein). When needed, holo-LF was prepared by saturating apo-LF with iron.

Effect of LF administered to animals was studied when pregnant or lactating Wistar rats (200 g) were injected intraperitoneally with apo-LF (50 mg per rat) or allowed drinking ad libitum water with apo- or holo-LF (5 mg/mL). Animals (pregnant or lactating rats, or suckling pups) were killed by decapitation under ether anesthesia and centrifuged ice-cold homogenates of their organs were studied either by Western blotting after electrophoresis in SDS-containing gel or by ELISA with anti-HIFs or anti-EPO.

Results: Provided that foreign apo-LF introduced to a rat by intraperitoneal injection achieves the maximum plasma concentration after 2 hours (4.65 µg/mL vs. 0.57 in controls), after which its concentration slowly declines (4.18 µg/mL after 4.5 hours), in our experiments, the pregnant rats received an intraperitoneal injection of bovine or human apo-LF (50 mg per animal) 48 to 72 hours prior to delivery, were killed after 18 hours, and their organs and whole embryos were studied. HIF-1a and HIF-2a were revealed by Western blotting with rabbit anti-HIFs in the brain, liver, heart, spleen, and placenta of females, but not in embryos. In the organs of control pregnant rats that did not receive apo-LF, no HIFs were revealed. Neither preparations of holo-LF (iron-saturated) had such an effect.

Similarly, EPO was revealed in the same organs of pregnant rats, but not in their embryos. Interestingly, no elevated content of EPO in sera of rats treated with apo- or holo-LF was observed.

The first group of lactating rats received an intraperitoneal injection of bovine apo-LF (100 mg per rat), and after 24 hours, three 10-day-old pups from each mother were withdrawn and examined.

Western blotting revealed HIF-1a, HIF-2a, and EPO in the brain, liver, and spleen of neonate rats. However, injections of holo-LF (100 mg per rat) had no such effect.

In the brain of sucklings Western blotting revealed EPO within the period between 3 and 25 hours from the intraperitoneal injection of apo-LF to their mothers.

The integrity of LF consumed by suckling pups was tested in an experiment when their organs were sampled 0.5, 1.5, 2.5, 3.5, and 5 hours after their mothers received human apo-LF. Studying the stomach content by SDS-electrophoresis and Western blotting showed that even after 5 hours the greater part of LF remained in its intact (nonproteolyzed) form, that is, having molecular mass 78 kDa. Minor amount of a fragment with Mr ca. 50 kDa was also observed.

Oral administration of apo-LF to lactating rats was organized so that animals were divided in three groups with unlimited access to (1) potable water, (2) water with 5 mg/mL apo-LF, or (3) with 5 mg/mL Fe-LF. Western blotting and ELISA showed that their pups received that protein (milk and stomach content examined). As a result, after 3 days in group (2) both the lactating rats had stabilized HIF-1a and HIF-2a in their brain, liver, and spleen, but also the sucklings had these two proteins in the same organs. Besides, EPO was revealed in the same organs of the pups. Holo-LF introduced to animals in group (3) had no such effect.

Discussion: Several important considerations generate from the above-described findings. First, KF is able to appear in the milk of lactating animals, no matter whether it is introduced per os or bypassing the digestive tract. Second, LF is likely unable to overcome the placental barrier. Third, only apo-LF looks capable of inducing the HIF-signaling system in the tissues of neonate animals, which is proven by the occurrence of HIF-1a, HIF-2a, and—most importantly—of EPO whose gene is known to be a target of these transcription factors. Finally, the protective role of LF for the neonatal organism was until recently believed being limited to its pronounced antibacterial effect. In view of the results here obtained, it can be suggested that apo-LF represents an important factor regulating the development of central nervous system and probably of other systems in a neonate mammal. This notion mostly refers to humans, since rats and some other mammalian species have no LF in their milk, which was one of the reasons why we used those animals in our experiments.

Conclusion: Apo-LF introduced to lactating rats either per os or intraperitoneally, induces HIF-signaling system in neonate animals, which is evidenced by occurrence of EPO in their tissues. Apo-LF seems therefore to be a potent regulatory factor in the neonatal development. It is important to consider its content in the breast milk and to develop approaches to a correct therapeutic use of LF provided externally.

Keywords: lactoferrin, erythropoietin, hypoxia-inducible factor-1 α, hypoxia-inducible factor-2 α