# Transferrin-dependent uptake and distribution of iron in osteoclast-like cells

## TL;DR

This study shows how iron is taken up and stored in osteoclast-like cells, revealing that iron-starved cells absorb more iron, and that transferrin helps retain iron in these cells.

## Contribution

The study reveals novel insights into iron uptake mechanisms and storage pools in osteoclasts under iron-deficient conditions.

## Key findings

- Iron-starved osteoclasts take up more iron compared to iron-saturated cells.
- Transferrin-mediated iron remains in cells, forming stable pools while non-transferrin iron is lost quickly.
- Iron does not traffic to ferritin or mitochondria in osteoclasts during the study period.

## Abstract

Iron is an essential micro component and is involved in numerous critical cellular processes and in energy production. While its roles in oxygen transport and in oxidative phosphorylation are well documented, it remains to be elucidated, whether iron modulates specific cellular processes in different organs. Iron deficiency has been found to lead to a decrease in the capacity of osteoclasts to dissolve amorphous calcium phosphate. Furthermore, levels of iron in the cellular environment led to significant changes in the levels of transcripts encoding iron transport proteins. Within the present study, the uptake of iron by osteoclasts and the kinetics of intracellular transport were analyzed. For this purpose, M-CSF (Macrophage-Colony Stimulating Factor) dependent non-adherent osteoclast progenitor cells were differentiated to osteoclasts in media containing M-CSF and RANKL (Receptor Activator of NF-κB Ligand). For the upregulation of iron transport capacity, media were supplemented with Deferoxamine, an iron complexor, rendering the cultures virtually iron-free. To analyze iron uptake by osteoclast like cells, holo-transferrin, loaded with 55Fe was added to the cells and iron uptake was quantitated in whole cell lysates and in fractionated cells. The data demonstrates that Deferoxamine-treated osteoclasts absorb higher quantities of iron as compared to untreated control cells. By density gradient centrifugation, cell associated iron can be separated into two major pools. Pool I represents non-transferrin associated iron in cytoplasmic fractions, while pool II contains transferrin/ transferrin receptor associated iron. Within 4 h of incubation in iron-deficient medium, pool I disappears, as does transferrin. Pool II iron and transferrin receptor, however, remain detectable. Furthermore, iron peaks did not associate with ferritin nor with mitochondria, demonstrating that these two mechanisms of iron storage did not become activated during the course of the study. The data thus demonstrates that iron uptake by osteoclasts can be modulated by exogenous iron and that cell associated iron forms either a labile iron pool of free iron that is lost within a short period of time or a vesicular pool of non-transferrin bound iron that remains stable over the experimental period. No further trafficking of iron into ferritin particles or mitochondria was detected.

•Exogenous iron levels modulate levels of transferrin receptors in osteoclasts.•Iron-starved osteoclasts are more efficient in taking up iron than saturated osteoclasts.•Iron taken up by osteoclasts through a transferrin-mediated mechanism remains in the cells.•No iron trafficking to mitochondria or ferritin particles was observed in osteoclasts.

Exogenous iron levels modulate levels of transferrin receptors in osteoclasts.

Iron-starved osteoclasts are more efficient in taking up iron than saturated osteoclasts.

Iron taken up by osteoclasts through a transferrin-mediated mechanism remains in the cells.

No iron trafficking to mitochondria or ferritin particles was observed in osteoclasts.