Heme-oxygenases during erythropoiesis in K562 and human bone marrow cells.

In mammalian cells, heme can be degraded by heme-oxygenases (HO). Heme-oxygenase 1 (HO-1) is known to be the heme inducible isoform, whereas heme-oxygenase 2 (HO-2) is the constitutive enzyme. Here we investigated the presence of HO during erythroid differentiation in human bone marrow erythroid precursors and K562 cells. HO-1 mRNA and protein expression levels were below limits of detection in K562 cells. Moreover, heme was unable to induce HO-1, at the protein and mRNA profiles. Surprisingly, HO-2 expression was inhibited upon incubation with heme. To evaluate the physiological relevance of these findings, we analyzed HO expression during normal erythropoiesis in human bone marrow. Erythroid precursors were characterized by lack of significant expression of HO-1 and by progressive reduction of HO-2 during differentiation. FLVCR expression, a recently described heme exporter found in erythroid precursors, was also analyzed. Interestingly, the disruption in the HO detoxification system was accompanied by a transient induction of FLVCR. It will be interesting to verify if the inhibition of HO expression, that we found, is preventing a futile cycle of concomitant heme synthesis and catabolism. We believe that a significant feature of erythropoiesis could be the replacement of heme breakdown by heme exportation, as a mechanism to prevent heme toxicity.

High expression of the cGMP-specific phosphodiesterase, PDE9A, in sickle cell disease (SCD) and the effects of its inhibition in erythroid cells and SCD neutrophils.

Modulation of intracellular cyclic guanosine monophosphate (cGMP) may characterize a therapeutic target for sickle cell disease (SCD); cGMP-dependent signalling may be important for erythroid foetal haemoglobin induction and exert anti-inflammatory functions in leucocytes. As the inhibition of phosphodiesterases (PDEs), which regulate intracellular cGMP, can result in tissue-specific elevation of cGMP, we studied the gene expressions of cGMP-specific PDEs (-1A, -5A and -9A) in the reticulocytes and neutrophils of healthy controls, steady-state SCD patients and SCD patients on hydroxycarbamide therapy (SCDHC). PDE9A gene expression was found in numerous cell types; however, high expression was found in neutrophils, reticulocytes, CD34(+)-derived erythroid cells and K562 erythroleukaemic cells, indicating a high haematopoietic cell expression. PDE9A gene expression was, however, significantly higher in the reticulocytes and neutrophils of SCD individuals, compared to control cells; Western blotting confirmed the production of PDE9A protein in SCD neutrophils and K562 cells. Inhibition of PDE9A enzyme with the specific inhibitor, BAY73-6691, significantly increased production of the gamma-globin gene (HBG) in K562 cells and reversed the increased adhesive properties of SCD neutrophils. Since elevation of haematopoietic intracellular cGMP may be beneficial in SCD, the relatively limited tissue distribution of PDE9A suggests that it could represent a novel drug target worthy of further study.