BACKGROUND

Low serum hepcidin levels provide a physiologic response to iron demand in patients with iron deficiency (ID). Based on a discovery of suppressed hepcidin expression by a cytokine named growth differentiation factor 15 (GDF15), it was hypothesized that GDF15 may suppress hepcidin expression in humans with ID due to blood loss.

STUDY DESIGN AND METHODS

To test this hypothesis, GDF15 and hepcidin levels were measured in peripheral blood from subjects with iron-deficient erythropoiesis before and after iron supplementation.

RESULTS

Iron variables and hepcidin levels were significantly suppressed in iron-deficient blood donors compared to healthy volunteers. However, ID was not associated with elevated serum levels of GDF15. Instead, iron-deficient subjects’ GDF15 levels were slightly lower than those measured in the control group of subjects (307 ± 90 and 386 ± 104 pg/mL, respectively). Additionally, GDF15 levels were not significantly altered by iron repletion.

CONCLUSIONS

ID due to blood loss is not associated with a significant change in serum levels of GDF15.

Background

MicroRNAs are ~22nt-long small non-coding RNAs that negatively regulate protein expression through mRNA degradation or translational repression in eukaryotic cells. Based upon their importance in regulating development and terminal differentiation in model systems, erythrocyte microRNA profiles were examined at birth and in adults to determine if changes in their abundance coincide with the developmental phenomenon of hemoglobin switching.

Methods

Expression profiling of microRNA was performed using total RNA from four adult peripheral blood samples compared to four cord blood samples after depletion of plasma, platelets, and nucleated cells. Labeled RNAs were hybridized to custom spotted arrays containing 474 human microRNA species (miRBase release 9.1). Total RNA from Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines provided a hybridization reference for all samples to generate microRNA abundance profile for each sample.

Results

Among 206 detected miRNAs, 79% of the microRNAs were present at equivalent levels in both cord and adult cells. By comparison, 37 microRNAs were up-regulated and 4 microRNAs were down-regulated in adult erythroid cells (fold change > 2; p < 0.01). Among the up-regulated subset, the let-7 miRNA family consistently demonstrated increased abundance in the adult samples by array-based analyses that were confirmed by quantitative PCR (4.5 to 18.4 fold increases in 6 of 8 let-7 miRNA). Profiling studies of messenger RNA (mRNA) in these cells additionally demonstrated down-regulation of ten let-7 target genes in the adult cells.

Conclusion

These data suggest that a consistent pattern of up-regulation among let-7 miRNA in circulating erythroid cells occurs in association with hemoglobin switching during the fetal-to-adult developmental transition in humans.

In vivo, inhibition of fetal hemoglobin (HbF) expression in humans around the time of birth causes the clinical manifestation of sickle cell and beta-thalassemia syndromes. Inhibition of HbF among cultured cells was recently described by the adenosine derivative molecule named SQ22536. Here, a primary cell culture model was utilized to further explore the inhibition of HbF by adenosine derivative molecules. SQ22536 demonstrated down-regulation of growth and HbF expression among erythroblasts cultured from fetal and adult human blood. The effects upon HbF were noted in a majority of cells, and quantitative PCR analysis demonstrated a transcriptional mechanism. Screening assays demonstrated two additional molecules named 5′-deoxy adenosine and 2′,3′-dideoxy adenosine had effects on HbF comparable to SQ22536. Other adenosine-derivative molecules, adenosine receptor binding ligands, and cAMP-signaling regulators failed to inhibit HbF in matched cultures. These results suggest structurally-related ribofuranose-substituted adenosine analogues act through an unknown mechanism to inhibit HbF expression in fetal and adult human erythroblasts.

Hembase (http://hembase.niddk.nih.gov) is an integrated browser and genome portal designed for web-based examination of the human erythroid transcriptome. To date, Hembase contains 15 752 entries from erythroblast Expressed Sequenced Tags (ESTs) and 380 referenced genes relevant for erythropoiesis. The database is organized to provide a cytogenetic band position, a unique name as well as a concise annotation for each entry. Search queries may be performed by name, keyword or cytogenetic location. Search results are linked to primary sequence data and three major human genome browsers for access to information considered current at the time of each search. Hembase provides interested scientists and clinical hematologists with a genome-based approach toward the study of erythroid biology.