The Cyclic GMP-Dependent Pathway Is Involved in the Mechanisms of Action of Hydroxyurea.

Abstract

Hydroxyurea (HU) is the standard therapeutic agent for patients with sickle cell disease (SCD). Administration of HU reduces the frequency of pain crisis, increases total hemoglobin levels, and decreases the numbers of reticulocytes, leukocytes, and platelets. However, the mechanism underlying these ameliorating effects of HU on the clinical expression of SCD patients still remains to be clarified. Several lines of evidence that were obtained from our laboratory and others suggest that HU exerts its molecular effects through the cyclic GMP (cGMP)-dependent pathway. To examine whether the cGMP-dependent pathway is involved in the molecular actions of HU, we generated mice that overexpress soluble guanylate cyclase (sGC), a key enzymes of the cGMP-dependent pathway and have an activated cGMP-dependent pathway in peripheral blood cells. Adult mice expressed the transgenes encoding sGC subunits in bone marrow cells, spleen cells and peripheral leukocytes. In these cells of transgenic sGC mice, intracellular cGMP levels were elevated and the phosphorylation levels of vasodilator-stimulated phosphoprotein, which is a substrate of protein kinase G of the cGMP-dependent pathway, were increased. These results demonstrated activation of the cGMP-dependent pathway in the sGC mice. Interestingly, high-level expression of sGC subunit transgenes resulted in down-regulation of endogenous mouse sGC mRNA expression, suggesting that mRNAs encoding sGC subunits are unstable in cells which have high cGMP levels. More interestingly, adult sGC mice had elevated levels of total hemoglobin, and decreased numbers of white blood cell counts, which is consistent with a clinical picture of SCD patients who are administered with HU. In vitro colony assays demonstrated that cGMP has positive and negative effects on the differentiation of erythroid- and myeloid-lineage cells, respectively. Furthermore, in sickle cell patients treated with HU, intracellular cGMP levels of red blood cells positively correlated with total hemoglobin levels, while leukocyte counts were inversely correlated with the cGMP levels of leukocytes. These results indicate the roles of the cGMP-dependent pathway at least in part in regulating blood cell counts in sickle cell patients. We previously reported that expression of the human gamma-globin gene is induced by activating the cGMP-dependent pathway (PNAS 98: 1847, 2001). Next, we examined whether expression of human gamma-globin, which is dormant in adult erythroid cells, is induced in sGC mice. We bred sGC mice with a YAC mouse carrying the entire human beta-globin gene locus. Although human gamma-globin was not detectable by HPLC in sGC/YAC mice, low but appreciable levels of human gamma-globin expression was induced following the activation of the cGMP-dependent pathway with protoporphyrin IX, which is an activator of sGC. This demonstrated a role of the pathway in inducing gamma-globin expression in adult erythroid cells. Collectively, these results shown in this study demonstrate that sGC mice exhibit hematologic characteristics that are similar to those of SCD patients on HU therapy, suggesting that the cGMP-dependent pathway is involved at least in part in the mechanisms of action of HU.