Excessive Isolated Erythrocytosis Linked to Induction of SCL/Tal1 and Erythropoietin Receptor Expression.

The JAK2 V617F mutation has been detected in more than 90% polycythemia vera patients and is associated with increased erythropoietin (EPO) sensitivity or EPO independence of erythroid progenitor cells during early erythropoiesis. To determine if other molecular lesions give rise to increased erythrocytosis and realizing that the JAK2 V617F mutation may not be the initiating event in polycythemia vera, we identified an individual with isolated, excessive erythrocytosis who showed erythroid precursor hypersensitivity to EPO; no mutations on sequencing genes for erythropoietin receptor (EPO-R), VHL, or HIF-1 IRE; normal serum EPO level; normal hemoglobin function and p50; normal cardiac, hepatic and pulmonary function; no JAK2 V617F mutation and no EPO independent BFU-E. While it was not certain that this individual’s erythrocytosis was lifelong, other relatives had increased hemoglobin levels. The cause of this individual’s increased erythropoiesis was investigated using EPO-stimulated cultures of hematopoietic progenitor cells isolated from peripheral blood. As with normal controls, cell numbers were not increased after 12 days of incubation with low levels of EPO (<0.1 U/ml). With 1U/ml of EPO, cell proliferation was similar to control early in erythropoiesis but increased markedly after 8 days so that by day 12 cell numbers were 3-fold greater than in control cultures with 95% benzidine positive cells compared with 76% for control and 82–87% for JAK2 V617F positive erythroid progenitor cells. Analyses of transcription factor expression revealed that induction of GATA-1 and down regulation of GATA-2 were similar to control. However, SCL/Tal1 and EKLF markedly increased beginning at day 8 to 10 and continued to rise during late erythropoiesis in contrast to control and JAK2 V617F positive cultures in which SCL/Tal1 and EKLF peaked at day 10 and decreased with late erythroid differentiation. Since increased beta-globin expression is concomitant with induction of EKLF, the rise in EKLF may account for the marked increase in benzidine positive cells from our subject. Moreover, expression of EPO-R followed the continuing rise in SCL/Tal1, increasing by 3.5 fold at day 12 compared with control cultures. This high EPO-R expression is consistent with the dramatic increase in cell proliferation during late erythropoiesis. Using forced expression of SCL/Tal1, reporter gene assay and gel mobility shift analysis in erythroid cells, we determined that SCL/Tal1 is able to bind to E-boxes located 3′ of the EPO-R proximal promoter and to activate transcription. Although EPO stimulation of erythroid progenitor cells activates EPO-R, we found that forced expression of EPO-R in primary erythroid progenitor cells in the presence of EPO increases expression of GATA-1 as well as SCL/Tal1 and EKLF, providing further evidence that specific increase in SCL/Tal1 but not GATA-1 must precede induction of EPO-R in this case of excessive erythrocytosis. These data demonstrate a link between high level induction of SCL/Tal1 expression and increased erythrocytosis and suggest a mechanism that contributes to increased erythropoietin sensitivity during late erythropoiesis.