We previously demonstrated that TIM-2 serves as a specific receptor for HFt in mouse cells 
. Results presented here demonstrate that iron bound to HFt can serve as a cellular iron source in mouse B cells or in mouse kidney cells that express TIM-2. Following uptake in TIM-2-positive cells, HFt traffics to endosomes and subsequently enters lysosomes (). This pathway differs from the classical pathway of iron delivery involving transferrin-mediated uptake of iron via TfR1, in which transferrin recycles to the cell surface following iron release in the endosome. Nevertheless, iron released from ferritin enters a metabolically available pool, and is able to upregulate synthesis of endogenous ferritin ().
Our work is consonant with other reports demonstrating both the ability of ferritin to enter the lysosome and to serve as an iron source. For example, recent work has shown that iron chelation can induce entry of cytosolic ferritin into lysosomes, where it undergoes degradation 
. Treatment of cells with cationic ferritin similarly led to delivery of ferritin to lysosomes, iron release, and induction of cytosolic ferritin synthesis 
. In human cells, TfR1 serves as a receptor for HFt 
, and may functionally substitute for TIM-2 
. Receptor-mediated uptake of HFt similarly directs ferritin to lysosomes in these cells 
. Collectively, these results imply the existence of a mechanism for the shuttling of iron from the lysosome to the cytosol. Candidate transport mechanisms that may mediate iron efflux out of the lysosome have recently been identified 
The relationship between TIM2 and Scara5, another recently identified ferritin receptor that functions in iron trafficking during development 
, is unclear. Unlike TIM-2, which selectively binds HFt, Scara5 binds LFt. Similar to our results with TIM-2, Scara5 mediates endocytosis of ferritin and ensuing delivery of iron to cells. It was suggested that Scara5 may functionally substitute for TfR1 in selected tissues of the kidney during development 
. The relationship between expression of Scara5 and TIM-2 has not been examined. In the developing mouse, Scara5 is expressed in stromal and capsular cells of the kidney as well as in the airway, developing aorta, muscle bundles and gonadal epithelia 
; in the adult mouse, Scara5 is expressed in epithelial cells of the testis, bladder, trachea, adrenal, skin, lung, and ovary 
. This spatial distribution demonstrates little overlap with TIM-2, which is expressed in adult B cells, bile duct epithelial cells, renal tubules and oligodendrocytes, suggesting that in the adult, expression patterns of these ferritin receptors are largely independent. However, further work will be required to determine whether Scara5 and TIM-2 exhibit any temporal or functional relationship.
Whether the TIM-2 pathway contributes in a substantial way to iron import remains to be determined. Our results demonstrate that TIM-2 can mediate uptake of ferritin and its associated iron; however, the amount of iron delivered to cells through this pathway will depend on the iron content of the ferritin, which can vary over more than 2 orders of magnitude 
It is possible that the iron content of ferritin may influence the ultimate cellular effect of TIM-2. For example, ferritin exhibits immunosuppressive effects, inhibiting the proliferation of T and B cells and the differentiation of myeloid cells 
. Since lymphocytes express TIM-2, it has been suggested that this anti-proliferative effect of ferritin could be mediated through TIM-2 
. We speculate that if ferritin contains little or no iron, it may serve as a signaling molecule to induce such anti-proliferative or apoptotic 
effects. Alternatively, if receptor-bound ferritin contains iron, it may serve as an iron source to support cell proliferation. Delivery of iron through ferritin has previously been proposed to occur in the developing kidney 
, and in macrophage-mediated delivery of ferritin-bound iron to erythroid precursors 
. Our results indicate that TIM-2 has the potential to function as an iron delivery mechanism, but further work will be required to elucidate the circumstances under which that mechanism is activated.
Although specific binding of ferritin to cell surfaces has been repeatedly documented 
, the source of ferritin that binds to TIM-2 and other ferritin receptors in vivo
remains unknown. In our experiments, cells were exposed to 4 nM ferritin, which is within the range found in the serum of patients with iron overload and other inflammatory conditions such as Stills disease, hemophagocytic syndrome, etc. 
. However, the majority of subunits in serum ferritin resemble ferritin L more closely than ferritin H 
, and since TIM-2 preferentially binds HFt, it is not clear that serum ferritin represents a likely source of ferritin ligand for TIM-2. Nevertheless, ferritin is a 24 subunit protein, and most natural ferritins are heteropolymers of both H and L subunits. Since the number of H subunits required for effective binding of TIM-2 has not yet been determined, ferritin proteins that contain a preponderance of L subunits may nonetheless bind to TIM-2. In addition, small amounts of H subunit-rich ferritin are present in the serum and can increase in certain pathological conditions 
. An alternative potential source of ferritin is local secretion. For example, the macrophage, which can serve as a source of circulating ferritin 
may also secrete ferritin locally 
, and this may serve as a paracrine source of ferritin for uptake by TIM-2. Indeed HFt was identified as a soluble ligand secreted by macrophage cell lines during the cloning of TIM-2 
. HFt is also released by hepatocytes 
. Future studies will be required to trace the physiological source of the HFt ligand.
Our results indicate that expression of TIM-2 is not increased by iron chelation (). This distinguishes TIM-2 from other iron uptake pathways. For example, TfR1, which mediates Tf-dependent uptake of iron, and DMT-1, which mediates transport of ferrous iron, are post-transcriptionally regulated by iron status 
. For these transporters, regulation is mediated by IRE elements on the 3′ end of the mRNA. Our sequence inspection demonstrated no obvious candidate IRE elements in the sequence of TIM-2. However, since IRE elements may exhibit substantial divergence in primary sequence 
, we cannot rule out the presence of a non-canonical IRE element in TIM-2. The lack of response of TIM-2 to iron chelation that we observed is different from observations in oligodenodrocytes, in which expression of TIM-2 was reported to be iron-regulated 
. Oligodendrocytes have no TfR1 to mediate iron uptake 
, may depend heavily on the TIM-2 pathway, and may have alternative mechanisms of regulation not found in other cell types. In contrast, in A20 cells, iron uptake is mediated by two pathways: the classical TfR1 pathway 
and the TIM-2 pathway described here. It is possible that uncoupled regulation of TfR1 and TIM-2 may permit differential activation of these pathways. For example, in A20 cells (and other cells that express both TfR1 and TIM-2), TIM-2 may serve as a backup pathway that can respond to environmental changes in HFt regardless of cellular iron status.