Ineffective Erythropoiesis Is the Major Cause of Microcytic Anemia in the TSAP6/Steap3 Null Mouse Model

STEAP3 (Six-Transmembrane Epithelial Antigen of Prostate 3) is the major ferrireductase in the erythroblast. Also named TSAP6 (Tumor Supressor Activated Pathway 6) after it had been found to play a role in cancer, its total ablation in the mouse leads to severe microcytic and hypochromic red cells with moderate anemia. The protein function appears conserved among mammals, as patients carrying a nonsense mutation in the TSAP6/STEAP3 gene have been reported with hypochromic anemia. Here, we investigated the mechanism leading to the anemia. In the present study, using the TSAP6/Steap3 knockout mice, we undertook a comprehensive hematologic characterization of the red cell compartment. Red cell indices derived using ADVIA 120 blood counter confirmed the hypochromic microcytic anemia phenotype with a marked reduction in the mean corpuscular volume (MCV; 21.5fL ± 1.3fL in knockout vs 45.2fL ± 1.5fL in wild-type X ± SD, p<0.001) and mean cell hemoglobin content (MCH; 5.8pg ± 0.1pg vs 14.5pg ± 0.1pg, p<0.001). The reticulocyte count was marginally elevated (5.7% ± 1.2% vs 2.6% ± 0.4%, p<0.05) indicating that the anemia is proliferative. By phase contrast microscopy and transmission electron microscopy, we observed marked anisocytosis as well as the presence of fragmenting erythrocytes. Consistent with this observation, we found by ektacytometry decreased membrane mechanical stability of knockout red cells. Interestingly, we were unable to document significant changes in the expression levels of the major skeletal and transmembrane proteins to account for this decrease in the membrane stability.

As defects in either the production or destruction of red cells can lead to anemia, we measured red cell survival and erythropoiesis in these mice. No differences in red cell survival could be documented using biotin labeled red cells implying that decreased survival cannot account for the anemia. However, when we monitored erythropoiesis using Ter119, CD44 and Forward Scatter (FSC) as markers of terminal differentiation, we found a decreased number of proerythroblasts in the bone marrow of TSAP6/Steap3^-/- animals (2.20% ± 0.49% vs 4.07% ± 0.77%, p<0.01). In addition, progression from the proerythroblastic to the orthochromatic stage was also affected, with accumulation of cells at the polychromatic stage. These findings imply that ineffective erythropoiesis is the dominant cause of anemia in these mice and that TSAP6/Steap3 may play a role during erythropoiesis beyond its role as ferrireductase. It is interesting to note that while the phenotype of microcytic red cells with decreased hemoglobin content in the TSAP6/Steap3 null mouse is similar to red cells in thalassemic and porphyria mice, there are distinct differences in the etiology of the hematologic phenotype.