Progressive iron overload is the most salient and ultimately fatal complication of ␤-thalassemia. However, little is known about the relationship among ineffective erythropoiesis (IE), the role of iron-regulatory genes, and tissue iron distribution in ␤-thalassemia. We analyzed tissue iron content and iron-regulatory gene expression in the liver, duodenum, spleen, bone marrow, kidney, and heart of mice up to 1 year old that exhibit levels of iron overload and anemia consistent with both ␤-thalassemia intermedia (th3/؉) and major (th3/th3). Here we show, for the first time, that tissue and cellular iron distribution are abnormal and different in th3/؉ and th3/th3 mice, and that transfusion therapy can rescue mice affected by ␤-thalassemia major and modify both the absorption and distribution of iron. Our study reveals that the degree of IE dictates tissue iron distribution and that IE and iron content regulate hepcidin (Hamp1) and other iron-regulatory genes such as Hfe and Cebpa. In young th3/؉ and th3/th3 mice, low Hamp1 levels are responsible for increased iron absorption. However, in 1-year-old th3/؉ animals, Hamp1 levels rise and it is rather the increase of ferroportin (Fpn1) that sustains iron accumulation, thus revealing a fundamental role of this iron transporter in the iron overload of ␤-thalasse- Introduction␤-Thalassemia is the most common congenital hemolytic anemia due to partial or complete lack of synthesis of ␤-globin chains. Cooley anemia, 1 also known as ␤-thalassemia major, is the most severe form of ␤-thalassemia, which is characterized by profound ineffective erythropoiesis (IE) requiring regular red blood cell (RBC) transfusions to sustain life. Transfusion therapy leads to excess iron accumulation in many organs resulting in tissue damage. Therefore, iron chelation is essential in the management of this otherwise fatal disease. 2 In ␤-thalassemia intermedia, in which a larger amount of ␤-globin chains are synthesized, the clinical picture is milder and the patients do not require frequent transfusions. However, progressive iron overload still occurs due to increased gastrointestinal (GI) iron absorption. [3][4][5] Studies in thalassemic patients showed that the rate of iron uptake from the GI tract is approximately 3 to 4 times greater than normal. 6 Ferrokinetic studies revealed that 75% to 90% of the iron in donor serum, labeled with 59 Fe and injected into healthy subjects, appeared in circulating red cells within 7 to 10 days. In some thalassemic patients, however, only 15% of the 59 Fe was incorporated into circulating erythrocytes. 7 This discrepancy was attributed to the fact that iron would be sequestered in those organs in which premature destruction of erythroid precursors occurs. In ␤-thalassemia, it has been suggested that 60% to 80% of erythroid precursors die in the marrow and extramedullary sites. [8][9][10] Therefore, in ␤-thalassemia erythropoietic organs such as the bone marrow (BM) in humans and the BM and spleen in mice would be expected to show the highest iron concen...

In ␤-thalassemia, the mechanism driving ineffective erythropoiesis (IE) is insufficiently understood. We analyzed mice affected by ␤-thalassemia and observed, unexpectedly, a relatively small increase in apoptosis of their erythroid cells compared with healthy mice. Therefore, we sought to determine whether IE could also be characterized by limited erythroid cell differentiation. In thalassemic mice, we observed that a greater than normal percentage of erythroid cells was in Sphase, exhibiting an erythroblast-like morphology. Thalassemic cells were associated with expression of cell cycle-promoting genes such as EpoR, Jak2, Cyclin-A, Cdk2, and Ki-67 and the antiapoptotic protein Bcl-X L . The cells also differentiated less than normal erythroid ones in vitro. To investigate whether Jak2 could be responsible for the limited cell differentiation, we administered a Jak2 inhibitor, TG101209, to healthy and thalassemic mice. Exposure to TG101209 dramatically decreased the spleen size but also affected anemia. Although our data do not exclude a role for apoptosis in IE, we propose that expansion of the erythroid pool followed by limited cell differentiation exacerbates IE in thalassemia. In addition, these results suggest that use of Jak2 inhibitors has the potential to profoundly change the management of this disorder. (Blood. 2008;112:875-885) Introduction ␤-Thalassemia, one of the most common congenital anemias, arises from partial or complete lack of ␤-globin synthesis. ␤-Thalassemia major, also known as Cooley anemia, 1 is the most severe form of this disease, and is characterized by ineffective erythropoiesis (IE) and extramedullary hematopoiesis (EMH), requiring regular blood transfusions to sustain life. [1][2][3][4][5] In ␤-thalassemia intermedia, where a larger amount of ␤-globin is synthesized, the clinical picture is milder and the patients do not require frequent transfusions. The ineffective production of red blood cells in both forms of the disease has been attributed to erythroid cell death during the maturation process mediated by apoptosis or hemolysis. It was proposed that accumulation of alpha-globin chains leads to the formation of aggregates, which impair erythroid maturation triggering apoptosis. [6][7][8][9][10][11][12][13] Ferrokinetic studies done in 1970 suggested that 60% to 80% of the erythroid precursors in ␤-thalassemia major die in the marrow or extramedullary sites. 14 However, several observations call into question the view that cell death is the only cause of IE in ␤-thalassemia.First, the number of apoptotic erythroid cells in thalassemic patients is low compared with that anticipated by ferrokinetic studies. 14,15 In fact, only 15% to 20% of bone marrow (BM) erythroid precursors (CD45 Ϫ /CD71 ϩ ) present apoptotic features in aspirates from affected patients. 6,8,16 Second, hemolytic markers in young ␤-thalassemic patients are normal or only slightly increased, unless the patients suffer from splenomegaly or the liver has been damaged by iron overload or viral infections. 17 Third...

HFE C282Y, the mutant protein associated with hereditary hemochromatosis (HH), fails to acquire the correct conformation in the endoplasmic reticulum (ER) and is targeted for degradation. We have recently shown that an active unfolded protein response (UPR) is present in the cells of patients with HH. Now, by using HEK 293T cells, we demonstrate that the stability of HFE C282Y is influenced by the UPR signaling pathway that promotes its degradation. Treatment of HFE C282Y-expressing cells with tauroursodeoxycholic acid (TUDCA), a bile acid derivative with chaperone properties, or with the chemical chaperone sodium 4-phenylbutyrate (4PBA) impeded the UPR activation. However, although TUDCA led to an increased stability of the mutant protein, 4PBA contributed to a more efficient disposal of HFE C282Y to the degradation route. Fluorescence microscopy and biochemical analysis of the subcellular localization of HFE revealed that a major portion of the C282Y mutant protein forms intracellular aggregates. Although neither TUDCA nor 4PBA restored the correct folding and intracellular trafficking of HFE C282Y, 4PBA prevented its aggregation. These data suggest that TUDCA hampers the UPR activation by acting directly on its signal transduction pathway, whereas 4PBA suppresses ER stress by chemically enhancing the ER capacity to cope with the expression of misfolded HFE, facilitating its degradation. Together, these data shed light on the molecular mechanisms involved in HFE C282Y-related HH and open new perspectives on the use of orally active chemical chaperones as a therapeutic approach for HH.

Calreticulin is an endoplasmic reticulum resident molecule known to be involved in the folding and assembly of major histocompatibility complex (MHC) class I molecules. In the present study, expression of calreticulin was analyzed in human peripheral blood T lymphocytes. Pulse-chase experiments in [35 S]methionine-labeled T cell blasts showed that calreticulin was associated with several proteins in the endoplasmic reticulum and suggested that it was expressed at the cell surface. Indeed, the 60-kDa calreticulin was labeled by cell surface biotinylation and precipitated from the surface of activated T cells together with a protein with an apparent molecular mass of 46 kDa. Cell surface expression of calreticulin by activated T lymphocytes was further confirmed by immunofluorescence and flow cytometry, studies that showed that both CD8؉ and CD4؉ T cells expressed calreticulin in the plasma membrane. Low amounts of cell surface calreticulin were detected in resting T lymphocytes. By sequential immunoprecipitation using the conformation independent monoclonal antibody HC-10, we provided evidence that the cell surface 46-kDa protein co-precipitated with calreticulin is unfolded MHC I. These results show for the first time that after T cell activation, significant amounts of calreticulin are expressed on the T cell surface, where they are found in physical association with a pool of ␤ 2 -free MHC class I molecules.Calreticulin is a highly conserved and widely tissue distributed calcium-binding protein with a C-terminal KDEL endoplasmic reticulum (ER) 1 retrieval sequence (1-3). However, calreticulin has also been found outside the ER, such as within the secretory granules of cytotoxic lymphocytes, the cell surface of melanoma cells and virus-infected fibroblasts, and the cytosol and nucleus of several cell types reviewed in Ref. 9). Given its lectin-like properties, calreticulin is considered to be an ER chaperone involved in the assembly and folding of nascent glycoproteins (10 -13).Mature MHC class I molecules are composites of a 44 -49-kDa polymorphic heavy chain and a 12-kDa light chain (␤ 2 m) complexed with a cytosolic-processed peptide that are expressed on the plasma membrane of almost every nucleated cell (14). A number of ER resident molecular chaperones, such as calnexin, BiP, and transporter-associated protein, are involved in the assembly of the different composites (15)(16)(17)(18). Recently, calreticulin has also been shown to function as a chaperone in the assembly and folding of MHC class I molecules in the ER (19 -22). Contrary to calnexin, calreticulin binds to MHC class I-␤ 2 m dimers and to transporter-associated protein via another chaperone, tapasin (19,21). After peptide loading and deglucosylation of N-linked glycans, calreticulin dissociates from the MHC class I-␤ 2 m dimers, thus allowing the final transport of mature MHC class I molecules to the cell surface (20).Recent studies on the biosynthesis of TcR⅐CD3 complexes suggest that some chaperones such as calnexin can escape the ER retent...