IntroductionErythropoiesis is mainly regulated by the kidney-produced hormone erythropoietin (Epo), which is absolutely required for the survival and proliferation of erythroid progenitors and their terminal differentiation to red cells. 1 The Epo receptor (EpoR) is a type 1 transmembrane protein that belongs to the class 1 cytokine receptor family. Its expression level in erythroid cells is low whatever the differentiation stage and even the most Epo-sensitive cells, colony-forming units erythroid (CFU-Es), or proerythroblasts express less than 1000 EpoRs per cell at their surface. 2 In the absence of Epo, EpoR is believed to be homodimeric, in which each dimer protein is constitutively associated with a Janus kinase-2 (Jak-2) tyrosine-kinase molecule. Epo binding modifies the organization of the receptor complex leading to Jak-2 activation. Association between Jak-2 and the EpoR occurs during the receptor maturation process, most likely before EpoR leaves the endoplasmic reticulum, and is essential for expression of the receptor at the cell surface. 3 However, the mechanisms that control the maturation and the transport of the EpoR to the cell surface remain largely unknown. Indeed, the number of cell surface EpoRs is not related to the synthesis level of this protein and most of these molecules accumulate in the endoplasmic reticulum in EpoRoverexpressing cells. 4 Both functional and direct evidence also suggests that unidentified proteins are associated with EpoR on the cell surface. In particular, chemical cross-linking of radiolabeled Epo to cell surface erythroid cells has frequently revealed the presence of 2 additional proteins with apparent molecular masses of 85 and 100 kDa in the EpoR complex. 2,5-8 Unfortunately, because of the low expression level of the EpoR, these proteins could never be identified up to now. The dramatic progress that has been recently introduced by the application of mass spectrometry methods to protein analysis led us to address the question of the identity of the EpoR-associated proteins.Here, we have purified the EpoR complex from UT7 erythroleukemic cells that express endogenous EpoRs to identify EpoRassociated proteins. Mass spectrometry analysis of the purified proteins revealed the presence of the type 2 transferrin receptor (TfR2) in the EpoR complex.Mammals express 2 transferrin receptors that share similar overall structures but possess specific functions. Most cells including erythroid precursors internalize iron from plasma diferric transferrin through the ubiquitously expressed transferrin receptor 1 (TfR1). A Tfr1 gene deletion is lethal in mice with severe anemia and is not compensated by TfR2. 9 In contrast, TfR2 expression is tissue-restricted with high expression in the liver 10 where it plays a key role in iron metabolism regulation. Indeed, TfR2 contributes to the adaptation of hepcidin production to the needs of the body by sensing the circulating iron bound to transferrin. 11 Familial inactivating or non-sense mutations in the TfR2 gene are responsible for...

Transferrin receptor 2 (TFR2) is a transmembrane protein that is mutated in

BackgroundUsefulness of iron chelation therapy in myelodysplastic patients is still under debate but many authors suggest its possible role in improving survival of low-risk myelodysplastic patients. Several reports have described an unexpected effect of iron chelators, such as an improvement in hemoglobin levels, in patients affected by myelodysplastic syndromes. Furthermore, the novel chelator deferasirox induces a similar improvement more rapidly. Nuclear factor-κB is a key regulator of many cellular processes and its impaired activity has been described in different myeloid malignancies including myelodysplastic syndromes. Design and MethodsWe evaluated deferasirox activity on nuclear factor-κB in myelodysplastic syndromes as a possible mechanism involved in hemoglobin improvement during in vivo treatment. Forty peripheral blood samples collected from myelodysplastic syndrome patients were incubated with 50 μM deferasirox for 18h. ResultsNuclear factor-κB activity dramatically decreased in samples showing high basal activity as well as in cell lines, whereas no similar behavior was observed with other iron chelators despite a similar reduction in reactive oxygen species levels. Additionally, ferric hydroxyquinoline incubation did not decrease deferasirox activity in K562 cells suggesting the mechanism of action of the drug is independent from cell iron deprivation by chelation. Finally, incubation with both etoposide and deferasirox induced an increase in K562 apoptotic rate. ConclusionsNuclear factor-κB inhibition by deferasirox is not seen from other chelators and is iron and reactive oxygen species scavenging independent. This could explain the hemoglobin improvement after in vivo treatment, such that our hypothesis needs to be validated in further prospective studies.Key words: iron chelation therapy, nuclear factor-κB, myelodysplastic symdrome. Haematologica 2010;95(8):1308-1316. doi:10.3324/haematol.2009 Deferasirox is a powerful NF-κB inhibitor in myelodysplastic cells and in leukemia cell lines acting independently from cell iron deprivation by chelation and reactive oxygen species scavenging Citation: Messa E, Carturan S, Maffè C, Pautasso M, Bracco E, Roetto A, Messa F, Arruga F, Defilippi I, Rosso V, Zanone C, Rotolo A, Greco E, Pellegrino RM, Alberti D, Saglio G, and Cilloni D. Deferasirox is a powerful NF-κB inhibitor in myelodysplastic cells and in leukemia cell lines acting independently from cell iron deprivation by chelation and reactive oxygen species scavenging.

© F e r r a t a S t o r t i F o u n d a t i o nvariants associate with iron and erythrocyte traits in different populations. [22][23][24][25][26][27] By studying Tmprss6 haploinsufficient mice 28 and hepcidin levels of normal individuals and the TMPRSS6 common single nucleotide polymorphism (rs855791) 29 we demonstrated that even a partial inability to modulate hepcidin influences iron parameters and, indirectly, erythropoiesis.The regulation of TMPRSS6 and its activity is incompletely understood: besides hypoxia, 30 iron and BMP6, through the BMP-SMAD pathway, induce TMPRSS6 expression, likely as a negative feedback loop to limit excessive increases of HAMP. 31 However, the regulation of TMPRSS6 in vivo according to iron needs remains to be clarified. A possible role of Tmprss6 in iron overload was demonstrated by Finberg et al. 32 who showed that Hfe -/-mice with complete loss of Tmprss6 revert from a phenotype of iron overload to one of iron-deficiency anemia with high Hamp levels. These findings suggest that HFE acts genetically upstream of TMPRSS6 in the modulation of the BMP-SMAD pathway and of HAMP expression. In analogy with these results and given the role of TFR2 in erythropoiesis 16 we wondered whether TFR2 is involved in the regulation of TMPRSS6. To answer this question, we back-crossed Tmprss6 -/-mice with animals with a complete deletion of Tfr2 (Tfr2 -/-) and analyzed the hematologic phenotype and the Bmp-Smad-Hamp pathway of the double mutant mice. Moreover, in order to discriminate between the hepatic and extra-hepatic functions of TFR2, we performed the same analysis in Tmprss6 -/-mice lacking Tfr2 specifically in the liver (Tfr2 LCKO ). 15 Methods Mouse modelsMice were maintained in the animal facility of the Department of Clinical and Biological Sciences, University of Turin (Italy) in accordance with European Union guidelines. Each study was approved by the Institutional Animal Care and Use Committee (IACUC) of the same institution.A Tmprss6 -/-mouse model on a mixed C57BL/6-Sv129 background was kindly provided by Prof. C. Lopez-Otin (University of Oviedo, Spain) and maintained by brother-sister mating for more than ten generations. Tfr2 -/-and Tfr2 LCKO mice on a pure 129S2 background were generated as previously described. 15 For the experimental work described we bred Tfr2 -/-or Tfr2 LCKO mice with Tmprss6 +/-mice and then intercrossed the F1 progeny to generate various genotype combinations (F2: wild-type, Tmprss6. Mice were given a standard diet (480 mg iron/Kg) and only male mice were analyzed when 10 weeks old. Blood was collected for hematologic analyses, transferrin saturation and erythropoietin levels. After sacrifice livers and spleens were dissected, weighed, and snap-frozen immediately for RNA analysis or dried for tissue iron quantification. Hematologic analysesBlood was obtained by retro-orbital puncture from anesthetized mice. Red blood cell and white blood cell counts, hemoglobin concentration, hematocrit and erythrocyte indices (mean corpuscular volume, mean cor...

Transferrin receptor 2 (Tfr2) is mainly expressed in the liver and controls iron homeostasis. Here, we identify Tfr2 as a regulator of bone homeostasis that inhibits bone formation. Mice lacking Tfr2 display increased bone mass and mineralization independent of iron homeostasis and hepatic Tfr2. Bone marrow transplantation experiments and studies of cell-specific Tfr2 knockout mice demonstrate that Tfr2 impairs BMP-p38MAPK signaling and decreases expression of the Wnt inhibitor sclerostin specifically in osteoblasts. Reactivation of MAPK or overexpression of sclerostin rescues skeletal abnormalities in Tfr2 knockout mice. We further show that the extracellular domain of Tfr2 binds BMPs and inhibits BMP-2-induced heterotopic ossification by acting as a decoy receptor. These data indicate that Tfr2 limits bone formation by modulating BMP signaling, possibly through direct interaction with BMP either as a receptor or as a co-receptor in a complex with other BMP receptors. Finally, the Tfr2 extracellular domain may be effective in the treatment of conditions associated with pathological bone formation.