Dissection of the Expression Patterns of Hydroxyurea Transporters during Erythropoiesis and Myelopoiesis

Background:Hydroxyurea (HU) is the only FDA approved drug for the treatment of moderate to severe sickle cell disease (SCD). It is becoming a standard of care for all SCD patients; clinical efficacy is attributed largely to enhanced expression of the γ-globin gene in erythroid progenitors. Dose-limiting toxicity of the drug in myeloid cells is believed to hinder its maximum effectiveness and utilization. Hitherto, the precise mechanisms that influence the on-target effects of HU in eyrthroid cells and the off-target cytotoxic effects in myeloid cells have not been defined.

Cell surface transporters regulating the intracellular accumulation of compounds exert a major influence on drug action. For instance, differential expression of transporters in specific cell types elicits variable responses. We have previously shown that the organic cation/carnitine transporter 1 (OCTN1) and urea transporter B (UTB) modulate intracellular accumulation of HU. Both transporters are highly expressed in the bone marrow however their role in the on- and off-target effects of HU has never been examined. In this study, we reasoned that variation in the expression of UTB and OCTN1 on hematopoietic progenitor cells (HPCs) may influence how individual lineages of HPCs respond to the drug.

Objective:In order to begin dissecting the role of OCTN1 and UTB transporters in modulating HU activity, the objective of the present study was to define lineage-specific expression patterns of UTB and OCTN1 in erythroid and myeloid progenitor cells during hematopoiesis. 

Methods: Human CD34+ HPCs isolated from peripheral blood were cultured in media containing erythropoietin or granulocyte-colony stimulating factor to induce erythropoiesis or myelopoiesis, respectively. Erythroid and myeloid differentiation was verified by flow cytometry and morphology. Specific populations of cells were isolated at defined time-points during differentiation, and gene expression for OCTN1 and UTB (encoded by SLC22A4 and SLC14A1respectively) was determined by quantitative RT-PCR.

Results: OCTN1 expression pattern was similar in erythroid and myeloid lineages but was variable during the course of differentiation. As cells differentiated from the common HPCs, OCTN1 expression was greatest in basophilic and polychromatic erythroblasts and promyelocytes. Expression increased 1.7- and 1.5-fold in erythroid and myeloid cells, respectively, compared to the common HPC. OCTN1 expression in late-stage precursor cells dropped to levels similar to those of common HPCs. Interestingly, UTB expression was 20-fold greater in othrochromatic erythroblasts and reticulocytes compared to common HPCs. However, the expression of this transporter decreased progressively in differentiating myeloid cells declining to a mean value 10-fold lower in late-stage myeloid band cells compared to the common HPCs. To examine whether HU treatment influences transporter expression, erythroblasts were treated with 100μM HU when OCTN1 and UTB expression was increased 10- and 20-fold, respectively (p<0.001). After 72 hours of HU exposure, γ-globin expression increased 2.5-fold (p<0.001) while OCTN1 expression increased 2-fold (p=0.01) compared to PBS-treated controls. UTB gene expression was significantly up regulated 1.5-fold (p<0.02) at 24 and 48 hours after HU treatment.  Taken together these data demonstrate differential expression patterns of OCTN1 and UTB in HPCs that are altered by HU.

Conclusions: These studies are the first to document HU transporter expression during erythroid and myeloid differentiation. We have shown that HU is a substrate for both OCTN1 and UTB, and we now demonstrate that their expression patterns are consistent with experimental evidence of HU response.  Optimal γ-globin induction is observed when early- to mid-stage progenitor cells are treated with HU and this coincides with significant increases in transporter expression. Preferential expression of UTB in erythroid progenitors suggests it may be a primary modulator of HU-mediated γ-globin induction with a lesser role in myeloid cytotoxicity. A detailed understanding of the expression patterns of OCTN1 and UTB in HPCs may help to refine the use of HU to improve its overall efficacy and utilization in SCD.