Soluble Guanylate Cyclase Modulators Effect Fetal Hemoglobin Levels through a Non-cGMP-Protein Kinase G Signaling Pathway
Authors: Sun Bin , Miller Valerie , Potoka Karin P , Nakamura Yukio , Gladwin Mark T , Bullock Grant C. 
Published: October 2019

Abstract
Sickle cell anemia results from a point mutation in both alleles of the β-globin gene. This homozygous mutation ultimately leads to a structural alteration of the hemoglobin protein that promotes polymerization of the mutant sickle hemoglobin tetramer (HbS) upon deoxygenation. HbS polymerization results in rigid, sickle-shaped RBCs with increased cell-to-cell adhesion properties. These sticky and rigid RBCs are prone to become trapped in small capillary networks leading to ischemia-reperfusion injury, endothelial damage and the hallmark pain crisis of sickle cell anemia. Neonates are protected from deoxy-HbS polymerization by high levels of fetal hemoglobin (HbF). HbF is composed of two α-globin and two γ-globin subunits(α2γ2), The γ-globin molecule cannot interact with deoxy-βS-globin polymers, which makes HbF an effective inhibitor of deoxy-HbS polymerization. The level of HbF required to reduce the symptoms of sickle cell anemia is 20-25%, but levels as low as 9% can prolong red cell survival. Treatment with hydroxyurea (HU) induces HbF and reduces the hematologic and clinical consequences of sickle cell anemia. Basal and inducible HbF levels are important in predicting the severity of sickle cell anemia and are highly phenotypically variable among patients, leading to varied responses to treatment. Patients are also variably susceptible to HU-induced cytopenias, which limits the use of HU in certain patients. HU is currently the only FDA-approved HbF-inducer for the treatment of sickle cell anemia and there is clearly a need for alternative HbF-inducers. Understanding the signaling pathways that regulate HbF induction will lead to novel therapeutic targets for sickle cell anemia. The soluble guanylate cyclase/cyclic guanosine monophosphate-dependent protein kinase (sGC/PKG) signaling pathway potentially links HU to the induction of HbF expression. In this study we investigated the direct role of sGC in HbF induction using novel pharmacologic modulators of sGC. Nitric oxide (NO) activates sGC by binding to the ferrous iron (Fe2+) in the active site heme moiety. Once activated, sGC converts GTP to cGMP, which in-turn activates PKG. Reactive oxygen species (ROS) oxidize the active site heme of sGC leading to NO-insensitivity. We tested the ability of a novel sGC activator, BAY 58-2667, to induce γ-globinin primary and immortalized (HUDEP-2) human erythroid progenitor cells. BAY 54-6544 binds to heme-free inactivated sGC to restore its guanylyl cyclase activity independent of NO. We also tested the ability of the sGC stimulator, BAY 41-2272, to induce γ-globinin primary and HUDEP-2 human erythroid progenitor cells. BAY 41-2272, binds to the ferrous iron at the active site of non-oxidized sGC to stimulate guanylyl cyclase activity in a synergistic manner with NO. We compared g-globin mRNA and protein expressionin the primary and immortalized human erythroid progenitors after treatment with different concentrations and combinations of BAY 54-6544, BAY 41-2667 and HU. We also evaluated g-globin induction in cellstreated with the pan-phosphodiesterase inhibitor IBMX and a synthetic cGMP analog. Although we see robust induction of cGMP and activation of PKG with all treatments, we only see significant induction of g-globin expression in the HU treated cells. This data suggests that the induction of HbF occurs through a non-sGC/PKG-dependent signaling pathway. These data demonstrate a very limited induction of γ-globin by BAY 54-6544 and BAY 41-2667 that appears to be disproportionate to, and independent of, cGMP/PKG signaling. These data also demonstrate, for the first time, that HU treatment of the immortalized HUDEP-2 cell line induces γ-globin expression more consistently than in primary erythroid progenitors.

Disclosures
No relevant conflicts of interest to declare.