Abstract
Iron overload and bony abnormalities cause considerable morbidity among patients with thalassemia syndromes. One possible explanation for this phenomenon is that proteins normally secreted into the marrow microenvironment during erythropoiesis are over-expressed in thalassemia patients due to expanded and ineffective erythropoiesis. We previously discovered that GDF15 is produced at very high levels in thalassemia patients and inhibits hepcidin expression. Transcriptome screens of erythroblasts were utilized here to identify twisted gastrulation (TWSG1) as a second candidate protein for further study. Quantitative PCR using the β-thalassemia murine model (Hbbth3/+ β-thalassemia intermedia mouse model, n=13; Hbbth3/th3 β-thalassemia major mouse model, n=5) revealed that splenic expression levels of Tsg (murine TWSG1) were significantly higher in thalassemia mice (Hbbth3/+, 2.2E02 ± 2.7E01 copies/ng RNA, p<0.01; Hbbth3/th3, 5.3E02 ± 6.8E01 copies/ng RNA, p<0.01) than among wild type mice (4.7E01 ± 2.4E01 copies/ng RNA, n=7). Bone marrow expression of Tsg was elevated (Hbbth3/+, 1.1E02 ± 3.2E01 copies/ng RNA, p=0.17; Hbbth3/th3, 1.3E02 ± 2.2E01 copies/ng RNA, p<0.05) compared with wild type mice (5.3E01 ± 2.5E01 copies/ng RNA). Tsg expression levels in the murine liver were also significantly higher (Hbbth3/+, 2.8E02 ± 4.6E01 copies/ng RNA, p<0.05; Hbbth3/th3, 3.9E02 ± 4.9E01 copies/ng RNA, p<0.01) than in wild type mice (1.5E02 ± 4.0E01 copies/ng RNA). These results suggest that expression of Tsg is up-regulated in the murine β-thalassemia model. By comparison, murine Tsg expression was up-regulated to a greater extent than GDF15 in the thalassemia mice. In addition to murine studies, human studies of TWSG1 were performed. Quantitative PCR using cultured human CD34+ cells demonstrated the highest-level expression of TWSG1 at the early stages of erythroblast differentiation (9.3E02 ± 1.4E02 copies/ng RNA). Preliminary ELISA analyses demonstrated statistically significant elevations in TWSG1 levels in serum from thalassemia patients (n=18, 463 ± 41 ng/ml) when compared to serum from healthy volunteers (n=10, 310 ± 45 ng/ml, p<0.05), but the relative increase in TWSG1 in humans was far less than previously reported for GDF15. To determine whether TWSG1 regulates hepcidin expression, assays were performed using a human hepatoma cell line (HuH-7). Unlike GDF15, TWSG1 did not directly affect hepcidin expression as measured by quantitative PCR in dosed assays (1–1,000 ng/ml TWSG1). However, TWSG1 was found to suppress hepcidin through an indirect mechanism involving bone morphogenic protein (BMP). BMPs regulate several tissue-specific processes including bone remodeling and induction of hepcidin expression in liver cells. In dosed-titrations, ≥100 ng/ml of TWSG1 resulted in a 50% reduction (p<0.05) in the BMP2 augmentation of hepcidin expression. These novel data suggest that TWSG1 is expressed at elevated levels in thalassemia and has the potential to affect BMP signaling processes including the regulation of hepcidin.
Internal cleavage and synergy with twisted gastrulation enhance BMP inhibition by BMPER
