Discovery of Growth Differentiation Factor 15 as an Erythroblast-Secreted Regulator of Hepcidin with Very High Level Expression in Patients with Thalassemia.

Abstract

Iron overload causes considerable morbidity associated with thalassemia due to inappropriate suppression of the iron regulator, hepcidin. One possible explanation for this phenomenon is that protein(s) that are normally secreted into the marrow microenvironment by erythroblasts become endocrine signals in patients with thalassemia due to the myeloproliferative nature of the disease. To test this hypothesis, progenitor and precursor cell transcriptional profiles were generated with Affymetrix GeneChip Human Genome U133 Plus 2.0 Arrays using primary erythroblasts cultured from 15 healthy human donors. For this study, informatic analyses were focused upon 54 members of the TGF–B/BMP superfamily. Among the subset of genes identified by this screen, evidence for high-level expression of a gene named growth differentiation factor 15 (GDF15) was discovered in the precursor cell profiles. Quantitative PCR, Western, and ELISA analyses confirmed expression and secretion of GDF15 during erythroblast maturation. GDF15 is an apoptosis-associated protein expressed primarily by the placenta. To determine whether GDF15 serves as a regulator of hepcidin expression, hepcidin expression assays were performed using a human hepatoma cell line (HuH-7). BMP2 and BMP4 were studied for comparison. Addition of BMP2 and BMP4 (range 10,000–500,000 pg/ml) resulted in dosed increases in hepcidin mRNA (5–30 fold). At the concentrations of GDF15 normally found in human blood (500 pg/ml), a 2-fold increase in the expression of hepcidin was measured compared to matched cultures containing no supplemental GDF15. However, GDF15 dosed to levels above 5,000 pg/ml resulted in a significant reduction in hepcidin expression. Next, plasma levels of GDF15 were measured in the peripheral blood of 162 donors (donor groups included healthy controls, sickle-cell syndromes, thalassemia syndromes, and other causes of anemia) to determine whether plasma GDF15 levels are dysregulated in thalassemia. Plasma samples from 21 hereditary hemochromatosis donors provided evidence that significantly elevated GDF15 expression is not associated with iron overload in the absence of erythroid pathology. Compared with mean plasma GDF15 levels of 536±222 pg/ml among the control samples, only patients with thalassemia intermedia, thalassemia major, and HbE-beta thalassemia showed significantly elevated plasma levels of GDF15 (9 donors; mean GDF15 24,600 pg/ml; range 8,980–75,100 pg/ml; p<0.01). Elevated ferritin levels (>370 ug/L) were noted in each of those patients regardless of their transfusion or chelation history. These novel findings suggest that GDF15 is secreted from human erythroblasts, released into the circulation at extremely high levels in thalassemia patients, and contributes to iron overloading in those patients by suppressing hepcidin expression.