Anemia and splenomegaly in cGKI-deficient mice

Abstract

To explore the functional significance of cGMP-dependent protein kinase type I (cGKI) in the regulation of erythrocyte survival, gene-targeted mice lacking cGKI were compared with their control littermates. By the age of 10 weeks, cGKI-deficient mice exhibited pronounced anemia and splenomegaly. Compared with control mice, the cGKI mutants had significantly lower red blood cell count, packed cell volume, and hemoglobin concentration. Anemia was associated with a higher reticulocyte number and an increase of plasma erythropoietin concentration. The spleens of cGKI mutant mice were massively enlarged and contained a higher fraction of Ter119 ؉ erythroid cells, whereas the relative proportion of leukocyte subpopulations was not changed. The Ter119 ؉ cGKI-deficient splenocytes showed a marked increase in annexin V binding, pointing to phosphatidylserine (PS) exposure at the outer membrane leaflet, a hallmark of suicidal erythrocyte death or eryptosis. Compared with control erythrocytes, cGKI-deficient erythrocytes exhibited in vitro a higher cytosolic Ca 2؉ concentration, a known trigger of eryptosis, and showed increased PS exposure, which was paralleled by a faster clearance in vivo. Together, these results identify a role of cGKI as mediator of erythrocyte survival and extend the emerging concept that cGMP/cGKI signaling has an antiapoptotic/prosurvival function in a number of cell types in vivo.apoptosis ͉ Ca2ϩ channels ͉ phosphatidylserine ͉ spleen N itric oxide (NO) has been shown to be a powerful regulator of cell survival (1, 2). Depending on the source or concentration of NO and the influence of additional regulators, NO may stimulate or inhibit apoptosis (3). NO exerts its effects in part through S-nitrosylation of target proteins. However, the effect of NO donors on Ca 2ϩ -induced phosphatidylserine (PS) exposure, a hallmark of apoptosis, could be mimicked by cGMP analogs (4), suggesting the involvement of soluble guanylyl cyclase, cGMP, and cGMP-dependent protein kinase type I (cGKI), a well known signaling cascade downstream of NO (5, 6).Recent studies indicated that erythroid cells possess a functional NO/cGMP pathway (7-9), which may be involved in the regulation of eryptosis, the suicidal death of erythrocytes (10). Erythrocyte cGMP production might also be stimulated by NO generated in the endothelium. The cGKI can also be activated independent of cGMP in response to oxidative stress (11). Eryptosis may follow osmotic shock, energy depletion, and oxidative stress (10), which activate Ca 2ϩ -permeable cation channels (12). Subsequent Ca 2ϩ entry leads to activation of Ca 2ϩ -sensitive K ϩ channels, exit of KCl with osmotically obliged water, and thus cell shrinkage (13). In addition, Ca 2ϩ entry triggers Ca 2ϩ -sensitive scrambling of the cell membrane (14, 15) with subsequent exposure of PS at the erythrocyte surface (12). Cell membrane scrambling may further be triggered by ceramide (16) and activation of protein kinase C (17). PS-exposing erythrocytes bind to PS receptors (18) and are recognized, ...

Hemin-induced suicidal erythrocyte death

“…Eryptosis is stimulated by increase in cytosolic Ca 2+ activity [5,11,12,19,41]. Phosphatidylserine-exposing erythrocytes are engulfed by macrophages and are thus rapidly eliminated from circulating blood [24,26,36]. Cytosolic Ca 2+ concentration is increased by activation of Ca 2+ -permeable cation channels [20,21,32,41].…”

Erythrocyte programmed cell death

“…Nitroxide is partially effective through activation of cGMP-dependent protein kinases (106)(107)(108). The mechanism is apparently effective under control conditions in vivo, as mice deficient of the cGMP-dependent protein kinase type I (cGKI) suffer from severe anaemia and splenomegaly (109).…”

Iron deficiency anemia in cyclic GMP kinase knockout mice

“…3,4 Immunocytochemistry of bone marrow indicated that macrophages but not erythroblasts express cGKI. 1 In contrast, it was found that erythroblasts and erythrocytes purified by immunomagnetic selection using labeled anti-Ter119 MicroBeads followed by magnetic cell sorting are cGKI positive.…”

“…Histological inspection indicated an increased erythrocyte content of the spleen, as reported by Föller and colleagues. 3 The spleen size and spleen weight were normalized by treatment with the PPI ( Figure 3A) in the cGKI -/-and cGKI RM mice. The iron content of the spleen was increased by either PPI treatment or iron injection, and was normalized by the com- …”

“…Therefore, it was suggested that the observed anemia was induced by the rapid erythrocyte clearance in the spleen. 3 We investigated the discrepancy concerning cGKI expression in erythrocytes/erythroblasts 1,3 using Western blots of purified erythrocytes/erythroblasts ( Figure 1). These experiments were carried out in three independent laboratories using different methods for purification of the erythrocytes.…”