Journal articles: 'Erythrocyte signaling pathways'

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Adderley, Shaquria P., Eileen A. Dufaux, Meera Sridharan, Elizabeth A. Bowles, Madelyn S. Hanson, Alan H. Stephenson, Mary L. Ellsworth, and Randy S. Sprague. "Iloprost- and isoproterenol-induced increases in cAMP are regulated by different phosphodiesterases in erythrocytes of both rabbits and humans." American Journal of Physiology-Heart and Circulatory Physiology 296, no. 5 (May 2009): H1617—H1624. http://dx.doi.org/10.1152/ajpheart.01226.2008.

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Activation of the G protein Gs results in increases in cAMP, a necessary step in the pathway for ATP release from rabbit and human erythrocytes. In all cells, the level of cAMP is the product of its synthesis by adenylyl cyclase and its hydrolysis by phosphodiesterases (PDEs). Both iloprost (Ilo), a PGI2 analog, and isoproterenol (Iso), a β-agonist, stimulate receptor-mediated increases in cAMP in rabbit and human erythrocytes. However, the specific PDEs associated with each of these signaling pathways in the erythrocyte have not been fully characterized. Previously, we reported that PDE3B is present in rabbit and human erythrocyte membranes and that PDE3 inhibitors potentiate Ilo-induced increases in cAMP. Here we report that inhibitors of either PDE2 or PDE4, erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) and rolipram, respectively, potentiate Iso-induced increases in cAMP in rabbit and human erythrocytes. Importantly, these inhibitors had no effect on cAMP increases associated with the incubation of erythrocytes with Ilo. In addition, we establish, for the first time, the presence of PDE2A protein in rabbit and human erythrocyte membranes. Finally, we determined that preincubation of human erythrocytes with EHNA and rolipram together potentiate Iso-induced ATP release, whereas preincubation with cilostazol enhances Ilo-induced release of ATP. These results are consistent with the hypothesis that, in rabbit and human erythrocytes, Ilo-induced increases in cAMP and ATP release are regulated by PDE3, whereas those associated with Iso are regulated by the activities of both PDE2 and PDE4. These studies demonstrate that PDE activity in these cells is localized to specific signaling pathways.

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Hanson, Madelyn S., Alan H. Stephenson, Elizabeth A. Bowles, Meera Sridharan, Shaquria Adderley, and Randy S. Sprague. "Phosphodiesterase 3 is present in rabbit and human erythrocytes and its inhibition potentiates iloprost-induced increases in cAMP." American Journal of Physiology-Heart and Circulatory Physiology 295, no. 2 (August 2008): H786—H793. http://dx.doi.org/10.1152/ajpheart.00349.2008.

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Increases in the second messenger cAMP are associated with receptor-mediated ATP release from erythrocytes. In other signaling pathways, cAMP-specific phosphodiesterases (PDEs) hydrolyze this second messenger and thereby limit its biological actions. Although rabbit and human erythrocytes possess adenylyl cyclase and synthesize cAMP, their PDE activity is poorly characterized. It was reported previously that the prostacyclin analog iloprost stimulated receptor-mediated increases in cAMP in rabbit and human erythrocytes. However, the PDEs that hydrolyze erythrocyte cAMP synthesized in response to iloprost were not identified. PDE3 inhibitors were reported to augment increases in cAMP stimulated by prostacyclin analogs in platelets and pulmonary artery smooth muscle cells. Additionally, PDE3 activity was identified in embryonic avian erythrocytes, but the presence of this PDE in mammalian erythrocytes has not been investigated. Here, using Western blot analysis, we determined that PDE3B is a component of rabbit and human erythrocyte membranes. In addition, we report that the preincubation of rabbit and human erythrocytes with the PDE3 inhibitors milrinone and cilostazol potentiates iloprost-induced increases in cAMP. In addition, cilostamide, the parent compound of cilostazol, potentiated iloprost-induced increases in cAMP in human erythrocytes. These findings demonstrate that PDE3B is present in rabbit and human erythrocytes and are consistent with the hypothesis that PDE3 activity regulates cAMP levels associated with a signaling pathway activated by iloprost in these cells.

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Zennadi, Rahima, Patrick C. Hines, Laura M. De Castro, Jean-Pierre Cartron, Leslie V. Parise, and Marilyn J. Telen. "Epinephrine acts through erythroid signaling pathways to activate sickle cell adhesion to endothelium via LW-αvβ3 interactions." Blood 104, no. 12 (December 1, 2004): 3774–81. http://dx.doi.org/10.1182/blood-2004-01-0042.

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The possible role of physiologic stress hormones in enhancing adhesion of sickle erythrocytes (SS RBCs) to endothelial cells (ECs) in sickle cell disease (SCD) has not been previously explored. We have now found that up-regulation of intracellular cyclic adenosine monophosphate (cAMP)–dependent protein kinase A (PKA) by epinephrine significantly increased sickle but not normal erythrocyte adhesion to both primary and immortalized ECs. Inhibition of serine/threonine phosphatases also enhanced sickle erythrocyte adhesion at least partially through a PKA-dependent mechanism. Adhesion was mediated through LW (intercellular adhesion molecule-4 [ICAM-4], CD242) blood group glycoprotein, and immunoprecipitation studies showed that LW on sickle but not on normal erythrocytes undergoes increased PKA-dependent serine phosphorylation as a result of activation. The major counter receptor for LW was identified as the αvβ3 integrin on ECs. These data suggest that adrenergic hormones such as epinephrine may initiate or exacerbate vaso-occlusion and thus contribute to the association of vaso-occlusive events with physiologic stress.

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Sun, Kaiqi, Yujin Zhang, Mikhail Bogdanov, William Dowhan, Modupe Idowu, Harinder S. Juneja, Michael R. Blackburn, Rodney E. Kellems, and Yang Xia. "Elevated Adenosine Signaling Via Adenosine A2B Receptor Induces Normal and Sickle Erythrocyte Sphingosine Kinase 1 Activity." Blood 124, no. 21 (December 6, 2014): 4067. http://dx.doi.org/10.1182/blood.v124.21.4067.4067.

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Abstract Sickle Cell Disease (SCD) is one of the most devastating hemolytic genetic disorders affecting millions worldwide. Erythrocytes possess high sphingosine kinase 1 (Sphk1) activity and are considered to be the major cell type for supplying plasma sphingosine-1-phosphate, a signaling lipid regulating multiple physiological and pathological functions. Recent studies revealed that erythrocyte SphK1 activity is up-regulated in sickle cell disease (SCD) and contributes to sickling and disease progression. However, how erythrocyte Sphk1 activity is regulated in SCD remains unknown. In an effort to identify specific factors and signaling pathways involved in regulation of erythrocyte SphK1 activities in SCD, we first chose to screen the effects of molecules known to induce SphK1 activities in other cell types and/or reported to be elevated in the circulation of SCD including tumor necrosis factor alpha (TNF-α), interleukin 1 (IL-1), endothelin 1 (ET-1), vascular endothelial growth factor (VEGF), S1P and adenosine, on the activities of SphK1 in cultured primary mouse normal erythrocytes. Among all of those molecules tested, we found that adenosine is a previously unidentified hypoxia inducible molecule directly inducing SphK1 activity in vitro in a time and dosage-dependent manner. Next, using four adenosine receptor deficient mice and pharmacological approaches, we determined that the A2B adenosine receptor (ADORA2B) is essential for adenosine-induced SphK1 activity in cultured primary mouse normal and sickle erythrocytes. Subsequently, we provided in vivo genetic evidence that adenosine deaminase (ADA)-deficiency leads to excess plasma adenosine and elevated erythrocyte SphK1 activity. Lowering adenosine by ADA enzyme therapy or genetic deletion of ADORA2B significantly reduced excess adenosine-induced erythrocyte SphK1 activity in ADA-deficient mice. Mechanistically, we revealed that PKA functions downstream of ADORA2B mediating ERK activation and subsequently underlying adenosine-induced SphK1 activities in cultured mouse erythrocytes. Finally, we conducted human translational studies and reported that adenosine signaling via ADORA2B directly increases SphK1 activity in cultured primary human normal and sickle erythrocytes in a PKA/ERK-dependent manner. Overall, our findings reveal a novel signaling network regulating erythrocyte SphK1 and highlight innovative mechanisms to control SphK1 activity in normal and sickle setting. Disclosures No relevant conflicts of interest to declare.

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Dinarelli, Simone, Marco Girasole, and Francesco Misiti. "Amyloid β peptide affects erythrocyte morphology: Role of intracellular signaling pathways." Clinical Hemorheology and Microcirculation 71, no. 4 (June 27, 2019): 437–49. http://dx.doi.org/10.3233/ch-199007.

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Stephan, Huber. "Editorial: Ion Channels of Mature Human Erythrocytes." Open Biology Journal 4, no. 1 (January 31, 2011): 1–2. http://dx.doi.org/10.2174/18750362010040100001.

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Ion channels in the plasma membrane serve multiple functions such as setting the membrane potential, adjusting the cell volume and the intracellular electrolyte concentrations or eliciting versatile cytosolic Ca2+ signals. Channel activities regulate many basic cellular processes. Among those are cell proliferation, migration, differentiation and apoptotic cell death. Although devoid of nuclei and mitochondria, mature mammalian erythrocytes maintain a full set of functional ion channels in their plasma membrane. This special issue of The Open Biology Journal focuses on ion channels in the membrane of mature human erythrocytes, their regulation and their putative functions. Mature human erythrocytes travel about 100 miles and circulate more than 100.000 times through the body during their normal life span of 120 ± 4 days. The most obvious task thereby is the transport of blood oxygen and carbon dioxide as well as buffering of the pH in the blood. These functions depend on carboanhydrase, hemoglobin and band 3 anion exchanger. The latter two are the most abundant proteins in the erythrocyte cytosol and membrane, respectively. Because of this high abundance of hemoglobin which accounts for 98% of the cytosol protein content and because of the substantial absence of intracellular organelles, mature human erythrocytes are commonly simplified to hemoglobincontaining sacks. In sharp contrast to this view, increasing numbers of proteins are identified in mature human erythrocytes. Among those are proteins that build up signaling cascades. Outside-in signaling via membrane receptors such as purinergic receptors, as well as inside-out signaling via release of, e.g., ATP has been reported (see the article of Duranton et al. in this special issue). Moreover, intracellular signaling molecules such as protein kinases, have been unequivocally demonstrated to be functional suggesting that mature human erythrocytes are endowed with complex signaling similar to nucleated cells. As an example, circulating erythrocytes are cellular sensors of the oxygen tension and mechanical stress. Decline in oxygen partial pressure results in release of ATP regulated, presumably, through Gs protein, adenylcyclase and protein kinase Adependent activation of the ABC transporter CFTR. The released ATP, in turn, triggers vessel dilatation via formation of nitric oxide by the endothelium. Nitric oxide also negatively feeds back to the erythrocyte where it down-regulates ATP release. Hence, by releasing ATP at decreased oxygen pressure erythrocytes adapt the microcirculation to the oxygen consumption. In addition to ATP release, signaling via adenylcyclase and protein kinase A activates a CFTR-associated anion channel in mature erythrocytes (reviewed by Bouyer et al. in this special issue). Moreover, mature human erythrocytes activate organic osmolyte and anion channels via ATP release and autocrine purinergic signaling which is reminiscent of the activation of osmolyte channels in nucleated cells upon cell swelling (reviewed by Duranton et al. in this special issue). This indicates that mature human erythrocytes functionally express ion channel-regulating pathways similar if not identical to those implemented in nucleated cells. Erythrocyte ion channels are also involved in sensing cellular stress. Shumilina and Huber report in this special issue that cellular stress such as oxidative stress activates ClC-2, a further type of anion channel in the erythrocyte membrane which participates in the cellular stress response. Being largely silent under resting conditions, erythrocyte channels may build up membrane conductances in the nS range upon various signals (such as oxidative stress). A strong activator of erythrocyte ion channel activity is the intraerythrocytic amplification of the malaria parasite Plasmodium falciparum. Accordingly, most of what we know about the electrophysiology of ClC-2, the CFTR-associated anion channel, and the organic osmolyte and anion channel in human erythrocytes came from whole-cell and single-channel recording in Plasmodium-infected cells. The data on ClC-2, the CFTR-associated channel and the osmolyte channel are discussed in this special issue by the articles of Shumilina and Huber, Bouyer et al., and Duranton et al., respectively. The fourth article in this special issue, the article of Lars Kästner, summarizes our current knowledge about the cation channels in erythrocytes. Mature human erythrocytes express KCa3.1 (Gardos) K+ channels, different types of nonselective cation channels including TRPC6 and NMDA receptors. In addition circumstantial evidence hints to the expression of the voltage-gated Ca2+ channel CaV2.1 in human erythrocytes. Besides the comprehensive catalogue of the erythrocyte channel types, the article of Lars Käster gives a rewarding compendium about the history of channel research in human erythrocytes. Taken together, this special issue provides an overview of the unexpected diversity of erythrocyte ion channels that endows these small enucleated cells with a toolkit for electrosignaling. This toolkit enables erythrocytes to quickly respond to internal or external stimuli with changes in cytosolic free Ca2+, de- or hyperpolarization of the membrane, cell swelling or shrinkage, or release of channel-permeable solutes such as ATP. Moreover, the ion channels are integral modules of complex programs such as oxygen-regulated ATP release. Malaria is one example how such programs are exploited by the parasite, to adapt the erythrocyte cytosol to its needs.

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Tamez, Pamela A., Hui Liu, Amittha Wickrema, and Kasturi Haldar. "P. falciparum Modulates Erythroblast Cell Gene Expression in Signaling and Erythrocyte Production Pathways." PLoS ONE 6, no. 5 (May 4, 2011): e19307. http://dx.doi.org/10.1371/journal.pone.0019307.

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Adebiyi, Morayo G., Jeanne M. Manalo, and Yang Xia. "Metabolomic and molecular insights into sickle cell disease and innovative therapies." Blood Advances 3, no. 8 (April 23, 2019): 1347–55. http://dx.doi.org/10.1182/bloodadvances.2018030619.

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Abstract Sickle cell disease (SCD) is an autosomal-recessive hemolytic disorder with high morbidity and mortality. The pathophysiology of SCD is characterized by the polymerization of deoxygenated intracellular sickle hemoglobin, which causes the sickling of erythrocytes. The recent development of metabolomics, the newest member of the “omics” family, has provided a powerful new research strategy to accurately measure functional phenotypes that are the net result of genomic, transcriptomic, and proteomic changes. Metabolomics changes respond faster to external stimuli than any other “ome” and are especially appropriate for surveilling the metabolic profile of erythrocytes. In this review, we summarize recent pioneering research that exploited cutting-edge metabolomics and state-of-the-art isotopically labeled nutrient flux analysis to monitor and trace intracellular metabolism in SCD mice and humans. Genetic, structural, biochemical, and molecular studies in mice and humans demonstrate unrecognized intracellular signaling pathways, including purinergic and sphingolipid signaling networks that promote hypoxic metabolic reprogramming by channeling glucose metabolism to glycolysis via the pentose phosphate pathway. In turn, this hypoxic metabolic reprogramming induces 2,3-bisphosphoglycerate production, deoxygenation of sickle hemoglobin, polymerization, and sickling. Additionally, we review the detrimental role of an impaired Lands’ cycle, which contributes to sickling, inflammation, and disease progression. Thus, metabolomic profiling allows us to identify the pathological role of adenosine signaling and S1P-mediated erythrocyte hypoxic metabolic reprogramming and hypoxia-induced impaired Lands' cycle in SCD. These findings further reveal that the inhibition of adenosine and S1P signaling cascade and the restoration of an imbalanced Lands' cycle have potent preclinical efficacy in counteracting sickling, inflammation, and disease progression.

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Koch, Marion, Katherine E. Wright, Oliver Otto, Maik Herbig, Nichole D. Salinas, Niraj H. Tolia, Timothy J. Satchwell, Jochen Guck, Nicholas J. Brooks, and Jake Baum. "Plasmodium falciparum erythrocyte-binding antigen 175 triggers a biophysical change in the red blood cell that facilitates invasion." Proceedings of the National Academy of Sciences 114, no. 16 (April 3, 2017): 4225–30. http://dx.doi.org/10.1073/pnas.1620843114.

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Invasion of the red blood cell (RBC) by the Plasmodium parasite defines the start of malaria disease pathogenesis. To date, experimental investigations into invasion have focused predominantly on the role of parasite adhesins or signaling pathways and the identity of binding receptors on the red cell surface. A potential role for signaling pathways within the erythrocyte, which might alter red cell biophysical properties to facilitate invasion, has largely been ignored. The parasite erythrocyte-binding antigen 175 (EBA175), a protein required for entry in most parasite strains, plays a key role by binding to glycophorin A (GPA) on the red cell surface, although the function of this binding interaction is unknown. Here, using real-time deformability cytometry and flicker spectroscopy to define biophysical properties of the erythrocyte, we show that EBA175 binding to GPA leads to an increase in the cytoskeletal tension of the red cell and a reduction in the bending modulus of the cell’s membrane. We isolate the changes in the cytoskeleton and membrane and show that reduction in the bending modulus is directly correlated with parasite invasion efficiency. These data strongly imply that the malaria parasite primes the erythrocyte surface through its binding antigens, altering the biophysical nature of the target cell and thus reducing a critical energy barrier to invasion. This finding would constitute a major change in our concept of malaria parasite invasion, suggesting it is, in fact, a balance between parasite and host cell physical forces working together to facilitate entry.

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Yipp, Bryan G., Stephen M. Robbins, Mary E. Resek, Dror I. Baruch, Sornchai Looareesuwan, and May Ho. "Src-family kinase signaling modulates the adhesion ofPlasmodium falciparum on human microvascular endothelium under flow." Blood 101, no. 7 (April 1, 2003): 2850–57. http://dx.doi.org/10.1182/blood-2002-09-2841.

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The pathogenicity of Plasmodium falciparum is due to the unique ability of infected erythrocytes (IRBCs) to adhere to vascular endothelium. We investigated whether adhesion of IRBCs to CD36, the major cytoadherence receptor on human dermal microvascular endothelial cells (HDMECs), induces intracellular signaling and regulates adhesion. A recombinant peptide corresponding to the minimal CD36-binding domain from P falciparum erythrocyte membrane protein 1 (PfEMP1), as well as an anti-CD36 monoclonal antibody (mAb) that inhibits IRBC binding, activated the mitogen-activated protein (MAP) kinase pathway that was dependent on Src-family kinase activity. Treatment of HDMECs with a Src-family kinase–selective inhibitor (PP1) inhibited adhesion of IRBCs in a flow-chamber assay by 72% (P < .001). More importantly, Src-family kinase activity was also required for cytoadherence to intact human microvessels in a human/severe combined immunodeficient (SCID) mouse model in vivo. The effect of PP1 could be mimicked by levamisole, a specific alkaline-phosphatase inhibitor. Firm adhesion to PP1-treated endothelium was restored by exogenous alkaline phosphatase. In contrast, inhibition of the extracellular signal–regulated kinase 1/2 (ERK 1/2) and p38 MAP kinase pathways had no immediate effect on IRBC adhesion. These results suggest a novel mechanism for the modulation of cytoadherence under flow conditions through a signaling pathway involving CD36, Src-family kinases, and an ectoalkaline phosphatase. Targeting endothelial ectoalkaline phosphatases and/or signaling molecules may constitute a novel therapeutic strategy against severe falciparum malaria.

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Lu, Yunzhe, Alicia Rivera, and Athar Chishti. "Dematin and Adducin Tether Sodium-Hydrogen Exchanger, NHE1, to Erythrocyte Membrane Cytoskeleton." Blood 128, no. 22 (December 2, 2016): 700. http://dx.doi.org/10.1182/blood.v128.22.700.700.

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Abstract Dematin is a critical component of the membrane junctional complex in red blood cells. It tethers the spectrin cytoskeleton proteins to the membrane and its genetic deletion in mice causes dissociation of the spectrin, actin and β-adducin from the membrane resulting in the collapse of the red blood cells (RBCs). As dematin lacks a transmembrane domain, it is still unclear how this critical component of the junctional complex is anchored to the RBC membrane. Our previous studies have shown that the multi-transmembrane glucose transporter-1 (GLUT1) interacts with dematin and β-adducin in human RBCs, suggesting a potential role for GLUT1 in recruiting dematin to the membrane. However, as mouse RBCs do not express a GLUT1 homologue, an equivalent membrane receptor for dematin and/or adducin in mice remains to be determined. Using multiple in vitro and in vivo biochemical assays, here we demonstrate that the ubiquitously expressed plasma membrane Na+/H+ exchanger, NHE1 (Slc9a1), is one of the receptors for dematin and β-adducin in mature mouse red blood cells. NHE1 directly interacts with the core domain of dematin. Moreover, the dematin headpiece domain mutant S381E, which binds to the core domain with a higher affinity than the wild type, abolished the biochemical interaction between dematin and NHE1. This observation suggests that NHE1 and dematin headpiece domain compete for the same binding site(s) on the core domain. Furthermore, this finding highlights a molecular mechanism whereby an intermolecular switch of dematin regulates its interaction with NHE1 by phosphorylation. Dematin and β-adducin directly interact with NHE1 at its membrane-proximal cytoplasmic domain, which in turn regulates NHE1 activity in response to growth factor stimuli and intracellular pH alterations. Accordingly,we observed an increased cellular sodium content in erythrocytes of dematin headpiece and adducin double knockout mice (DAKO), suggesting a higher NHE1 activity in DAKO erythrocytes. Unlike GLUT1, NHE1 is expressed in both mouse and human RBCs. Thus, our results provide a novel mechanism for linking NHE1 to membrane skeleton and multiple cell signaling pathways through dematin and adducin (Figure 1). Since NHE1 is one of the major regulators of intracellular pH and hypertonic stress, our findings raise the possibility that the dematin-adducin-NHE1 complex may modulate these functions in RBCs as well as in other cell types with broad impact on the regulation of the actin cytoskeleton and cell migration. Figure 1 Dematin and adducin link erythrocyte junctional complex to membrane via multiple receptors. A, WT RBC cytoskeleton. B, Mutant (DAKO) RBC cytoskeleton. Images show a grossly deranged membrane skeleton in DAKO as compared to wild type. Red arrows show enlarged pores and yellow arrows indicate the presence of aggregates. Bars correspond to 0.2 µM. C, Schematic diagram of dematin, adducin, and NHE1 linking the complex to multiple signaling pathways. D, Proposed new model of the erythrocyte junctional complex. Figure 1. Dematin and adducin link erythrocyte junctional complex to membrane via multiple receptors. A, WT RBC cytoskeleton. B, Mutant (DAKO) RBC cytoskeleton. Images show a grossly deranged membrane skeleton in DAKO as compared to wild type. Red arrows show enlarged pores and yellow arrows indicate the presence of aggregates. Bars correspond to 0.2 µM. C, Schematic diagram of dematin, adducin, and NHE1 linking the complex to multiple signaling pathways. D, Proposed new model of the erythrocyte junctional complex. Disclosures No relevant conflicts of interest to declare.

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Tripathi, Abhai K., Wei Sha, Vladimir Shulaev, Monique F. Stins, and David J. Sullivan. "Plasmodium falciparum–infected erythrocytes induce NF-κB regulated inflammatory pathways in human cerebral endothelium." Blood 114, no. 19 (November 5, 2009): 4243–52. http://dx.doi.org/10.1182/blood-2009-06-226415.

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Abstract Cerebral malaria is a severe multifactorial condition associated with the interaction of high numbers of infected erythrocytes to human brain endothelium without invasion into the brain. The result is coma and seizures with death in more than 20% of cases. Because the brain endothelium is at the interface of these processes, we investigated the global gene responses of human brain endothelium after the interaction with Plasmodium falciparum–infected erythrocytes with either high- or low-binding phenotypes. The most significantly up-regulated transcripts were found in gene ontology groups comprising the immune response, apoptosis and antiapoptosis, inflammatory response, cell-cell signaling, and signal transduction and nuclear factor κB (NF-κB) activation cascade. The proinflammatory NF-κB pathway was central to the regulation of the P falciparum–modulated endothelium transcriptome. The proinflammatory molecules, for example, CCL20, CXCL1, CXCL2, IL-6, and IL-8, were increased more than 100-fold, suggesting an important role of blood-brain barrier (BBB) endothelium in the innate defense during P falciparum–infected erythrocyte (Pf-IRBC) sequestration. However, some of these diffusible molecules could have reversible effects on brain tissue and thus on neurologic function. The inflammatory pathways were validated by direct measurement of proteins in brain endothelial supernatants. This study delineates the strong inflammatory component of human brain endothelium contributing to cerebral malaria.

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Wang, Xiaoyu, Linda S. Marton, Bryce K. A. Weir, and R. Loch Macdonald. "Immediate early gene expression in vascular smooth-muscle cells synergistically induced by hemolysate components." Journal of Neurosurgery 90, no. 6 (June 1999): 1083–90. http://dx.doi.org/10.3171/jns.1999.90.6.1083.

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Object. Vasospasm after subarachnoid hemorrhage is associated with changes in modulators of vascular tone in the arterial wall and is related to the presence of erythrocyte hemolysate in the subarachnoid space. The purpose of this study was to determine the compounds in erythrocyte hemolysate that are responsible for changing smooth-muscle cell gene expression.Methods. Rat aorta smooth-muscle cells were exposed to erythrocyte hemolysate in vitro and the effects on immediate early gene messenger (m)RNA levels were determined by competitive reverse transcriptase—polymerase chain reaction.Message levels for c-fos, jun B, and c-jun were increased in the presence of hemolysate, reaching maximum expression between 30 and 60 minutes, whereas the level of jun D mRNA was unaffected. Increasing doses of hemolysate caused greater expression of c-fos and jun B, but not c-jun. Adenosine triphosphate and hemoglobin, possible spasmogens present in hemolysate, caused much smaller and more rapid increases in c-fos expression than whole hemolysate. Size fractionation showed that all of the c-fos mRNA—inducing activity of hemolysate was recovered with molecules greater than 6 kD. Following separation of hemolysate proteins by hydrophobic interaction chromatography, only one of the three fractions had partial activity. Recombining the three fractions, however, yielded greater c-fos activation than any combination of two.Conclusions. Multiple high-molecular-weight components present in erythrocytes have synergistic effects on gene expression in smooth-muscle cells. The differences in patterns of gene induction suggest that multiple signaling pathways are activated.

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Yuan, Mengxia, Qi He, Zhiyong Long, Xiaofei Zhu, Wang Xiang, Yonghe Wu, and Shibin Lin. "Exploring the Pharmacological Mechanism of Liuwei Dihuang Decoction for Diabetic Retinopathy: A Systematic Biological Strategy-Based Research." Evidence-Based Complementary and Alternative Medicine 2021 (August 2, 2021): 1–20. http://dx.doi.org/10.1155/2021/5544518.

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Objective. To explore the pharmacological mechanism of Liuwei Dihuang decoction (LDD) for diabetic retinopathy (DR). Methods. The potential targets of LDD were predicted by PharmMapper. GeneCards and other databases were used to collect DR genes. Cytoscape was used to construct and analyze network DR and LDD’s network, and DAVID was used for Gene Ontology (GO) and pathway enrichment analysis. Finally, animal experiments were carried out to verify the results of systematic pharmacology. Results. Five networks were constructed and analyzed: (1) diabetic retinopathy genes’ PPI network; (2) compound-compound target network of LDD; (3) LDD-DR PPI network; (4) compound-known target network of LDD; (5) LDD known target-DR PPI network. Several DR and treatment-related targets, clusters, signaling pathways, and biological processes were found. Animal experiments found that LDD can improve the histopathological changes of the retina. LDD can also increase erythrocyte filtration rate and decrease the platelet adhesion rate ( P < 0.05 ) and decrease MDA and TXB2 ( P < 0.05 ). Compared with the model group, the retinal VEGF and HIF-1α expression in the LDD group decreased significantly ( P < 0.05 ). Conclusion. The therapeutic effect of LDD on DR may be achieved by interfering with the biological processes (such as response to insulin, glucose homeostasis, and regulation of angiogenesis) and signaling pathways (such as insulin, VEGF, HIF-1, and ErbB signaling pathway) related to the development of DR that was found in this research.

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Diwan, Abhinav, Andrew G. Koesters, Amy M. Odley, Theodosia A. Kalfa, and Gerald W. Dorn. "Enhanced Erythroblast Mobilization in Nix-Deficient Mice Confers Resistance to Phenylhydrazine-Induced Anemia Despite Accelerated Erythrocyte Turnover." Blood 110, no. 11 (November 16, 2007): 3659. http://dx.doi.org/10.1182/blood.v110.11.3659.3659.

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Abstract Steady-state and dynamic regulation of erythrocyte production occurs by altering the balance of cell-survival versus apoptosis signaling in maturing erythroblasts. Previously, the pro-apoptotic factor Nix was identified as a critical death signal in normal erythropoietic homeostasis, acting in opposition to erythroblast-survival signaling by erythropoietin and Bcl-xl. However, the role of Nix in stress-erythropoiesis is not known. Here, by comparing the consequences of erythropoietin administration, acute phenylhydrazine-induced anemia, and aging in wild-type and Nix-deficient mice, we show that complete absence of Nix, or its genetic ablation specifically in hematopoietic cells, mimics the effects of erythropoietin (Epo). Both Nix ablation and Epo treatment increase early erythroblasts in spleen and bone marrow and increase the number of circulating reticulocytes, while maintaining a pool of mature erythroblasts as an “erythropoietic reserve”. As compared with WT, Nix null mice develop polycythemia more rapidly after Epo treatment, consistent with enhanced sensitivity to erythropoietin observed in vitro. After phenylhydrazine administration, anemia in Nix-deficient mice is less severe and recovers more rapidly than in WT mice, despite lower endogenous Epo levels. Anemic stress depletes mature erythroblasts in both WT and Nix null mice, but Nix null mice with basal erythroblastosis are resistant to anemic stress. These findings show that Nix null mice have greatly expanded erythroblast reserve and respond normally to Epo- and anemia-stimulated induction of erythropoiesis. However, the hematocrits of young adult Nix null mice are not elevated, and these mice paradoxically develop anemia as they age with decreased hemoglobin content (10g/dl) and hematocrit (36%; at 80±3 weeks of age) compared to WT mice (13g/dl and 46%; 82±5 weeks of age), inspite of persistent erythoblastosis observed in the bone marrow and spleen. Nix null erythrocytes, which are macrocytic and exhibit membrane abnormalities typically seen in immature cells or with accelerated erythropoiesis, demonstrate shorter life span with a half life of 5.2±0.6 days in the peripheral circulation by in vivo biotin labeling (as compared with a half life of 11.7±0.9 days in WT), and increased osmotic fragility as compared with normal erythrocytes. This suggests that production and release of large numbers of reticulocytes in Nix null mice can decrease erythrocyte survival. To rule out a non-hematopoietic consequence of Nix ablation that contributes to or causes increased erythrocyte fragility and in vivo consumption, such as primary hypersplenism, we undertook Tie2-Cre mediated conditional Nix gene ablation. Nixfl/fl + Tie2-Cre mice (hematopoietic-cell specific Nix null) develop erythroblastosis with splenomegaly, reticulocytosis, absence of polycythemia and increased erythrocyte fragility; suggesting that erythroblastosis and accelerated erythrocyte turnover are a primary consequence of Nix ablation in hematopoietic cells. Hence, dis-inhibition of erythropoietin-mediated erythroblast survival pathways by Nix ablation enhances steady-state and stress-mediated erythropoiesis.

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Parisi, Sarah, Carlo Finelli, Antonietta Fazio, Alessia De Stefano, Sara Mongiorgi, Stefano Ratti, Alessandra Cappellini, et al. "Clinical and Molecular Insights in Erythropoiesis Regulation of Signal Transduction Pathways in Myelodysplastic Syndromes and β-Thalassemia." International Journal of Molecular Sciences 22, no. 2 (January 15, 2021): 827. http://dx.doi.org/10.3390/ijms22020827.

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Erythropoiesis regulation is essential in normal physiology and pathology, particularly in myelodysplastic syndromes (MDS) and β-thalassemia. Several signaling transduction processes, including those regulated by inositides, are implicated in erythropoiesis, and the latest MDS or β-thalassemia preclinical and clinical studies are now based on their regulation. Among others, the main pathways involved are those regulated by transforming growth factor (TGF)-β, which negatively regulates erythrocyte differentiation and maturation, and erythropoietin (EPO), which acts on the early-stage erythropoiesis. Also small mother against decapentaplegic (SMAD) signaling molecules play a role in pathology, and activin receptor ligand traps are being investigated for future clinical applications. Even inositide-dependent signaling, which is important in the regulation of cell proliferation and differentiation, is specifically associated with erythropoiesis, with phospholipase C (PLC) and phosphatidylinositol 3-kinase (PI3K) as key players that are becoming increasingly important as new promising therapeutic targets. Additionally, Roxadustat, a new erythropoiesis stimulating agent targeting hypoxia inducible factor (HIF), is under clinical development. Here, we review the role and function of the above-mentioned signaling pathways, and we describe the state of the art and new perspectives of erythropoiesis regulation in MDS and β-thalassemia.

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Kalfa, Theodosia A., Alex George, Suvarnamala Pushkaran, Xiuli An, Clinton H. Joiner, Narla Mohandas, and Yi Zheng. "Sickle Erythrocytes Have Increased Adducin Phosphorylation and Increased ROS Production Mediated by Signaling Pathways Involving Protein Kinase C and Rac GTPases." Blood 114, no. 22 (November 20, 2009): 901. http://dx.doi.org/10.1182/blood.v114.22.901.901.

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Abstract Abstract 901 Although sickle hemoglobin (HbSS) polymerization in hypoxic conditions is the trigger for the sickling deformation of red blood cells (RBC) containing HbSS, subsequent changes in the cytoskeleton and the mechanisms by which reversibly sickled cells become irreversibly malformed have not been fully elucidated. Actin oligomers are a significant structural component of the erythrocyte cytoskeleton. The small Rho GTPases Rac1 and Rac2 regulate actin structures and mediate reactive oxygen species (ROS) production via NADPH oxidase in a variety of cells (Schwartz.M., J.Cell Science 2004). Deficiency of Rac1 and Rac2 GTPases in mice disrupts the normal hexagonal organization of the RBC cytoskeleton and reduces erythrocyte deformability. This is associated with increased phosphorylation of adducin (an F-actin capping protein) at Ser-726, a domain-target of protein kinase C (PKC) (Kalfa et al, Blood 2006). We evaluated the cytoskeleton phosphorylation changes in RBCs from patients with HbSS and from control subjects with hemoglobin AA (HbAA) by immunoblotting with phospho-specific antibodies. We found that phosphorylation of adducin at Ser-726 and of band 4.1 at Ser-331 were significantly increased in the erythrocyte cytoskeleton of HbSS-RBCs. This was associated with elevated PKC activity in these cells, compared with normal RBCs. ROS concentration was also elevated in HbSS-RBCs by 150-250% compared to that in HbAA-RBC, as determined by flow cytometry using 5-(and 6-)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate, a peroxide-sensitive probe. Inhibition of PKC or Rac activity in HbSS-RBCs by small molecule-inhibitors, calphostin and NSC23766 respectively, resulted in decreased ROS production in a dose-dependent fashion, while PKC activation by phorbol 12-myristate 13-acetate (PMA) increased ROS production, implicating a PKC-Rac axis in erythrocyte ROS production. Additionally, Rac inhibition resulted in dose-dependent increase of adducin phosphorylation while PKC inhibition had the opposite effect. Preincubation of HbSS RBCs with calphostin appeared to decrease the subsequent susceptibility of these cells to sickling under hypoxic conditions, suggesting a role for PKC activity in the transition from normal to sickle morphology, likely through a combination of effects on volume regulation and membrane stability of the cells. Our results imply that aberrant cytoskeleton protein phosphorylation and ROS production in HbSS-RBCs, mediated by signaling pathways involving PKC and Rac GTPases, may contribute to membrane changes induced by cell sickling. Elucidation of these pathways may identify new therapeutic targets in sickle cell disease. Disclosures: No relevant conflicts of interest to declare.

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Gregory, Richard C., Ning Jiang, Kazuo Todokoro, Jill Crouse, Robert E. Pacifici, and Don M. Wojchowski. "Erythropoietin Receptor and STAT5-Specific Pathways Promote SKT6 Cell Hemoglobinization." Blood 92, no. 4 (August 15, 1998): 1104–18. http://dx.doi.org/10.1182/blood.v92.4.1104.

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Abstract Erythrocyte production in mammals is known to depend on the exposure of committed progenitor cells to the glycoprotein hormone erythropoietin (Epo). In chimeric mice, gene disruption experiments have demonstrated a critical role for Epo signaling in development beyond the erythroid colony-forming unit (CFU-e) stage. However, whether this might include the possible Epo-specific induction of red blood cell differentiation events is largely unresolved. To address this issue, mechanisms of induced globin expression in Epo-responsive SKT6 cells have been investigated. Chimeric receptors containing an epidermal growth factor (EGF) receptor extracellular domain and varied Epo receptor cytoplasmic domains first were expressed stably at physiological levels in SKT6 cells, and their activities in mediating induced hemoglobinization were assayed. While activity was exerted by a full-length chimera (EE483), truncation to remove 7 of 8 carboxyl-terminal tyrosine sites (EE372) markedly enhanced differentiation signaling. Moreover, mutation of a STAT5 binding site in this construct (EE372-Y343F) inhibited induced globin expression and SKT6 cell hemoglobinization, as did the ectopic expression of dominant-negative forms of STAT5 in parental SKT6 cells. As in normal CFU-e, SKT6 cells also were shown to express functional receptors for stem cell factor (SCF). To further define possible specific requirements for differentiation signaling, effects of SCF on SKT6 cell hemoglobinization were tested. Interestingly, SCF not only failed to promote globin expression but inhibited this Epo-induced event in a dose-dependent, STAT5-independent fashion. Thus, effects of Epo on globin expression may depend specifically on STAT5-dependent events, and SCF normally may function to attenuate terminal differentiation while promoting CFU-e expansion. © 1998 by The American Society of Hematology.

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Gregory, Richard C., Ning Jiang, Kazuo Todokoro, Jill Crouse, Robert E. Pacifici, and Don M. Wojchowski. "Erythropoietin Receptor and STAT5-Specific Pathways Promote SKT6 Cell Hemoglobinization." Blood 92, no. 4 (August 15, 1998): 1104–18. http://dx.doi.org/10.1182/blood.v92.4.1104.416k38_1104_1118.

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Erythrocyte production in mammals is known to depend on the exposure of committed progenitor cells to the glycoprotein hormone erythropoietin (Epo). In chimeric mice, gene disruption experiments have demonstrated a critical role for Epo signaling in development beyond the erythroid colony-forming unit (CFU-e) stage. However, whether this might include the possible Epo-specific induction of red blood cell differentiation events is largely unresolved. To address this issue, mechanisms of induced globin expression in Epo-responsive SKT6 cells have been investigated. Chimeric receptors containing an epidermal growth factor (EGF) receptor extracellular domain and varied Epo receptor cytoplasmic domains first were expressed stably at physiological levels in SKT6 cells, and their activities in mediating induced hemoglobinization were assayed. While activity was exerted by a full-length chimera (EE483), truncation to remove 7 of 8 carboxyl-terminal tyrosine sites (EE372) markedly enhanced differentiation signaling. Moreover, mutation of a STAT5 binding site in this construct (EE372-Y343F) inhibited induced globin expression and SKT6 cell hemoglobinization, as did the ectopic expression of dominant-negative forms of STAT5 in parental SKT6 cells. As in normal CFU-e, SKT6 cells also were shown to express functional receptors for stem cell factor (SCF). To further define possible specific requirements for differentiation signaling, effects of SCF on SKT6 cell hemoglobinization were tested. Interestingly, SCF not only failed to promote globin expression but inhibited this Epo-induced event in a dose-dependent, STAT5-independent fashion. Thus, effects of Epo on globin expression may depend specifically on STAT5-dependent events, and SCF normally may function to attenuate terminal differentiation while promoting CFU-e expansion. © 1998 by The American Society of Hematology.

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Bonomini, Mario, Luisa Pieroni, Maurizio Ronci, Vittorio Sirolli, and Andrea Urbani. "Blood Cell Proteomics in Chronic Kidney Disease." Open Urology & Nephrology Journal 11, no. 1 (July 31, 2018): 28–38. http://dx.doi.org/10.2174/1874303x01811010028.

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Background: The uremic syndrome mimes a systemic poisoning with the retention of numerous compounds which are normally removed by the kidney. The study of proteins and peptides, or proteomics, represents an important field of research for the investigation of blood and blood diseases. Methods and Materials: We focused our review on the results of proteomic investigations on blood cells of uremic patients with particular regard to the study of red blood cells, platelets, and monocytes. Results: In literature there are few, preliminary studies on platelets and monocytes while the knowledge on uremic erythrocytes is much wider. Proteomic investigations showed that erythrocyte membrane proteome of uremic patients, differs significantly from the proteome of healthy subjects, being characterized by an extensive remodeling which may influence visco-elastic properties of RBC such as deformability and involve diverse molecular pathways driving red blood cell signaling and removal. Conclusion: Proteomic technologies emerged as a useful tool in defining and characterizing both physiological and disease processes being able, among others, to give important insights into uremic anemia.

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Forester, Craig M., Zhen Shi, Maria Barna, and Davide Ruggero. "A Tailor-Made Protein Synthesis Program Drives Erythroid Development and Disease." Blood 126, no. 23 (December 3, 2015): 3581. http://dx.doi.org/10.1182/blood.v126.23.3581.3581.

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Abstract Erythropoiesis constitutes the largest demand on the hematopoietic system due to its extraordinary production on a daily basis. The erythroid proteome requires an integration of multiple external cues to coordinate programs of differentiation as well as maintenance of erythroid precursors. The biomedical relevance of this critical process is underscored by recent findings showing impaired ribosome function in an entire class of clinical disorders with severe impairments in erythroid differentiation, known as ribosomopathies, which remain poorly understood. One of the main signaling pathways controlling post-transcriptional gene expression during erythropoiesis is the mTOR pathway. mTOR activation downstream of SCF/Epo in erythroid progenitors controls the activity of the major cap-binding protein eIF4E. However, the functional role of eIF4E during erythropoiesis and protein synthesis control in this cell type remains unexplored. Here we show that eIF4E activity, through mTOR-dependent phosphorylation of its inhibitory protein 4EBP1, unexpectedly undergoes a dynamic switch between early erythroid precursor populations and during terminal erythrocyte maturation, where eIF4E becomes progressively silenced. Employing a unique eIF4E transgenic mouse model, we strikingly show that overexpression of eIF4E in the bone marrow compartment results in an early accumulation of erythrocyte precursors and a block in erythrocyte differentiation. Surprisingly, this new role of eIF4E in erythropoiesis is independent from control of global protein synthesis but instead may promote a specialized program of translation control that is customized for erythroid cell function. Employing state of the art unbiased proteomics, our work is uncovering distinct networks of proteins, whose expression levels are controlled by eIF4E dosage during specific phases of erythrocyte maturation. Together, our research highlights a novel molecular program linking exquisite regulation of eIF4E activity to specialized translational control underlying erythroid development, providing unprecedented insight into the etiology of erythroid dysfunction in ribosomopathies. Disclosures No relevant conflicts of interest to declare.

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Bruns, Ingmar, Ron-Patrick Cadeddu, Ines Brückmann, Sebastian Buest, Julia Fröbel, Thomas Schroeder, Stefanie Geyh, et al. "The Disease-Related Bone Marrow Microenvironment Alters Hematopoietic Stem and Progenitor Function in Multiple Myeloma Patients." Blood 118, no. 21 (November 18, 2011): 2898. http://dx.doi.org/10.1182/blood.v118.21.2898.2898.

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Abstract Abstract 2898 Multiple myeloma (MM) patients often suffer from hematopoietic impairment already at the time of diagnosis with anemia as the prevailing symptom. Given the overt affection of the bone marrow in MM patients by the invasion of malignant plasma cells, we hypothesized that hematopoietic insufficiency in these patients may originate from a functional impairment of hematopoietic stem and progenitor cells. Quantitative analysis of BM CD34+ HSPC cell subsets from MM patients and age-matched healthy donors showed a significant decline of all HSPC subsets including hematopoietic stem cells, common myeloid and lymphoid progenitors, granulocyte-macrophage progenitors and megakaryocyte-erythrocyte progenitors in MM patients. The greatest diminution was observed in megakaryocyte-erythrocyte progenitors (MEP) which were 4.9-fold reduced in comparison to healthy donors. Transcriptional analyses of CD34+ HSPC subsets revealed a significant deregulation of signaling pathways that was particularly striking for TGF beta signaling and suggested increased activation of this signaling pathway. Immunhistochemical staining of phosphorylated smad2, the downstream mediator of TGF receptor I kinase activation, in bone marrow sections and immunoblotting of purified CD34+ HSPC of MM patients confirmed the overactivation of TGF beta signaling. On a functional level, we observed significantly reduced long-term self-renewal and clonogenic growth, particularly of the erythroid precursors BFU-E and CFU-E, in CD34+ HSPC of MM patients which could be restored by inhibition of TGF beta signaling. Proliferation and cell cycle analyses revealed a significantly decreased proliferation activity in CD34+ HSPC and, particularly, MEP. Again, this was reversible after inhibition of TGF beta signaling. In addition, the transcriptional analyses showed disturbance of pathways involved in the adhesion and migration of HSPC and the gene encoding for the principal hyaluronan receptor CD44 throughout the HSPC subsets. This was corroborated by immunofluorescence imaging of CD44 on HSPC subsets showing a marked downregulation in the patients' cells. In line, the adhesion of CD34+ HSPC subsets to hyaluronan and their migration towards SDF-1 was significantly inhibited. Subsequent xenotransplantation of CD34+ HSPC from MM patients and healthy donors into myeloma-free recipients revealed even increased long-term engraftment of CD34+ HSPC obtained from MM patients and normal differentiation capacities suggesting that the observed functional alterations in fact depend on the MM-related bone marrow microenvironment. Our data show that hematopoietic impairment in patients with multiple myeloma originates, at least in part, from functional alterations of hematopoietic stem and progenitor cells. These alterations seem to depend on the disease-related changes of the bone marrow microenvironment. Currently, experiments are underway to elucidate in more detail the role of the microenvironment and the responsible structures for the impairment of HSPC in MM patients. These data will be presented. Disclosures: Kobbe: Celgene: Consultancy, Research Funding; Ortho Biotec: Consultancy.

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Erdman, Laura, Gabriela Cosio, Samir N. Patel, Sergio Grinstein, and Kevin C. Kain. "Innate inflammatory and phagocytic responses to Plasmodium falciparum: linked processes or molecularly discrete pathways?s." Clinical & Investigative Medicine 30, no. 4 (August 1, 2007): 81. http://dx.doi.org/10.25011/cim.v30i4.2851.

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Background: Effective innate immune responses are important for control of malaria blood-stage infection and in preventing progression to severe malaria in non-immune individuals. Key innate defenses include a tightly regulated inflammatory response and host clearance of parasitized erythrocytes (PE). Pattern recognition receptors on macrophages mediate these processes: parasite glycosylphosphatidylinositol (GPI) activates TLR2 to induce inflammation – an excess of which is associated with severe malaria – while scavenger receptor CD36 mediates non-opsonic uptake of PEs. Both pathways are potential therapeutic targets, but it is unclear whether they are interdependent. Findings in other systems implicate CD36 in inflammation and TLR2 in phagocytosis, and recent evidence indicates that CD36 and TLR2 can directly cooperate. Methods: We investigated whether the innate inflammatory and phagocytic responses of macrophages to P. falciparum are separable: does CD36-mediated PE internalization have inflammatory outcomes, and does TLR2 regulate PE uptake? CD36-mediated endocytosis failed to induce TNFα production. As a more representative model of innate PE uptake, α-CD36 EBABs (Erythrocyte-Biotin-Avidin-Biotinylated antibody) were generated; macrophages internalized EBABs in a CD36-specific manner via a signaling pathway similar to that of PE uptake. Results: Compared to controls, neither PE nor EBAB internalization induced TNFα release, indicating that the inflammatory consequences of CD36 engagement are ligand dependent. Regarding TLR2 regulation of PE uptake, wild type and Tlr2-/- macrophages showed no differences in EBAB or PE uptake. Pre-treatment of macrophages with P. falciparum GPI enhanced EBAB internalization, but this effect was CD36-independent and generalizable to other TLR ligands. Conclusions: These results suggest that innate inflammatory and phagocytic responses of macrophages to malaria are discrete. Thus, therapeutic augmentation of CD36-mediated PE uptake should not exacerbate inflammation, nor should inhibition of the TLR2 pathway compromise CD36-mediated PE clearance. The role of TLR-enhanced internalization in malaria will be further examined.

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Liu, Qingqing, Jiahua Liu, Yaya Du, Weiyan Guo, Jie Mi, and Yanyan Guo. "Network Pharmacology and Molecular Docking Analysis to Explore the Mechanism of Huaiqihuang-Mediated Alleviation of Henoch–Schönlein Purpura Nephritis." BioMed Research International 2022 (November 4, 2022): 1–13. http://dx.doi.org/10.1155/2022/2798217.

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Objective. Henoch–Schönlein purpura nephritis (HSPN) is considered a major cause of chronic renal failure and is the most common secondary glomerular disease in children. Huaiqihuang (HQH), a traditional Chinese herbal formula, exhibits therapeutic effects against HSPN in clinical practice. However, the potential molecular targets and mechanisms underlying HSPN treatment remain unclear. Methods. By constructing a protein-protein interaction (PPI) network, core targets related to HQH and HSPN were identified. Gene Ontology enrichment and Kyoto Encyclopedia of Genes and Genomes pathways were analyzed to identify the main pathways related to HSPN based on the core targets. To screen the main active ingredients of HQH against HSPN, an ingredient-target-pathway network was constructed using the top 10 main pathways associated with HSPN. Then, molecular docking was performed to explore the interactions and binding patterns between molecules and proteins. Results. Clinical data showed that HQH combined with conventional medicine significantly reduced 24-hour urine protein excretion, urine microalbumin levels, and erythrocyte counts in the urine sediment of HSPN patients. By constructing PPI models, 15 potential core targets were identified. The top 10 main pathways showed higher enrichment ratios, including the cytokine–cytokine receptor interaction and signaling pathways related to NOD-like receptor, IL-17, etc. Through the ingredient-target-pathway network and molecular docking, we revealed that five active ingredients of HQH had good affinities with three core targets, AKT1, MMP9, and SERPINE1, which may be vital in treating HSPN. Conclusions. The study preliminarily explored the active ingredients, targets, and pathways involved in HQH therapy for HSPN. The mechanism of HQH therapy may be attributed to the modulation of inflammatory response, immune response, and oxidative stress. Combined with clinical data, our results indicate that HQH is highly effective in treating HSPN.

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Bruns, Ingmar, Ron-Patrick Cadeddu, Ines Brueckmann, Julia Fröbel, Stefanie Geyh, Sebastian Büst, Johannes C. Fischer, et al. "Multiple myeloma–related deregulation of bone marrow–derived CD34+ hematopoietic stem and progenitor cells." Blood 120, no. 13 (September 27, 2012): 2620–30. http://dx.doi.org/10.1182/blood-2011-04-347484.

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Abstract Multiple myeloma (MM) is a clonal plasma cell disorder frequently accompanied by hematopoietic impairment. We show that hematopoietic stem and progenitor cells (HSPCs), in particular megakaryocyte-erythrocyte progenitors, are diminished in the BM of MM patients. Genomic profiling of HSPC subsets revealed deregulations of signaling cascades, most notably TGFβ signaling, and pathways involved in cytoskeletal organization, migration, adhesion, and cell-cycle regulation in the patients. Functionally, proliferation, colony formation, and long-term self-renewal were impaired as a consequence of activated TGFβ signaling. In accordance, TGFβ levels in the BM extracellular fluid were elevated and mesenchymal stromal cells (MSCs) had a reduced capacity to support long-term hematopoiesis of HSPCs that completely recovered on blockade of TGFβ signaling. Furthermore, we found defective actin assembly and down-regulation of the adhesion receptor CD44 in MM HSPCs functionally reflected by impaired migration and adhesion. Still, transplantation into myeloma-free NOG mice revealed even enhanced engraftment and normal differentiation capacities of MM HSPCs, which underlines that functional impairment of HSPCs depends on MM-related microenvironmental cues and is reversible. Taken together, these data implicate that hematopoietic suppression in MM emerges from the HSPCs as a result of MM-related microenvironmental alterations.

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Tutaeva, Victoria, J. Will Thompson, Matthew W. Foster, M. Arthur Moseley, Stephen P. Holly, Martha Delahunty, Leslie Parise V., and Marilyn J. Telen. "S-Nitrosylation of Rap1 and Relationship to Rap1 Activity and Disease Status In SCD." Blood 116, no. 21 (November 19, 2010): 2662. http://dx.doi.org/10.1182/blood.v116.21.2662.2662.

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Abstract Abstract 2662 Red cell adhesion appears to be a critical contributor to vaso-occlusion in sickle cell disease (SCD). We have been interested in intra-erythrocytic signaling events that can affect sickle red cell (SS RBC) adhesion and thereby stimulate vaso-occlusion. One such agonist-inducible signaling pathway is that of the β2 adrenergic receptor. β2 adrenergic receptor signaling pathways, involving both protein kinase A and the small guanosine triphosphatase Rap1, have been shown to affect multiple RBC adhesion receptors. Therefore, we sought to determine whether specific post-translational modifications of RBC membrane and intracellular proteins were associated with the signaling pathways affecting adhesion. Protein S-nitrosylation, a post-translational modification of cysteine thiol by nitric oxide (NO), is one of several post-translational protein modifications that can regulate diverse cell signaling pathways. Given the many reports of abnormal NO biology in SCD, we investigated how S-nitrosylation of erythrocyte proteins affects molecules involved in regulation of SS RBC adhesion. First, we identified RBC membrane proteins that underwent S-nitrosylation in RBCs from healthy donors (n=2) and SCD patients (n=6; three patients treated with hydroxyurea [HU] and three without HU treatment). Highly purified RBC membrane ghosts were pretreated with or without 0.05 mM CysNO (to promote S-nitrosocysteine formation) for 10 min, and S-nitrosylated proteins were isolated using the resin-assisted capture (SNO-RAC) method (Forrester et al. 2009). Captured S-nitrosylated proteins were visualized by SDS-PAGE, and protein identification was accomplished via an in-situ digestion of the bead-bound proteins, release of the residual bead-bound peptides, and analysis using nanoscale capillary liquid chromatography coupled to high resolution, accurate mass tandem mass spectrometry (LC/MS/MS). A number of proteins underwent S-nitrosylation in both normal and SS RBCs. Among these, MS analysis indicated that Rap1, which has previously been shown to affect SS RBC adhesion to laminin, underwent S-nitrosylation much more extensively in SS than in normal RBCs. These data were confirmed by SNO-RAC followed by immunoblotting. Using a RalGDS Ras-binding domain pull-down assay, we further quantitated the amount of detectable activated Rap1 in highly purified preparations of normal and SS RBCs (lacking either contaminating leukocytes or platelets), before and after RBC loading with NO. Active Rap1 was undetectable or minimally present in RBCs from healthy donors (n=6) but easily detectable in all SS RBCs (n=4). Loading of RBCs with NO in these same samples led to an increased amount of detectable activated Rap1 in both normal and SS RBCs. Finally, we found that patients admitted for pain episodes (n=3) had higher levels of Rap1 nitrosylation and activity than patients in steady state (n=5). In summary, we have found that Rap1, a protein that acts downstream of adrenergic signaling in RBCs, undergoes S-nitrosylation and that this modification is associated with increased Rap1 activity. Most importantly, SS RBCs contain significantly more S-nitrosylated Rap1 and activated Rap1 than do normal RBCs, and this difference in content of active Rap1 is more pronounced in patients with ongoing vaso-occlusion. Disclosures: Telen: GlycoMimetics: Consultancy.

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Zennadi, Rahima, Erin J. Whalen, Erik J. Soderblom, Susan C. Alexander, J. Will Thompson, Laura G. Dubois, M. Arthur Moseley, and Marilyn J. Telen. "Erythrocyte plasma membrane–bound ERK1/2 activation promotes ICAM-4–mediated sickle red cell adhesion to endothelium." Blood 119, no. 5 (February 2, 2012): 1217–27. http://dx.doi.org/10.1182/blood-2011-03-344440.

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Abstract The core pathology of sickle cell disease (SCD) starts with the erythrocyte (RBC). Aberration in MAPK/ERK1/2 signaling, which can regulate cell adhesion, occurs in diverse pathologies. Because RBCs contain abundant ERK1/2, we predicted that ERK1/2 is functional in sickle (SS) RBCs and promotes adherence, a hallmark of SCD. ERK1/2 remained active in SS but not normal RBCs. β2-adrenergic receptor stimulation by epinephrine can enhance ERK1/2 activity only in SS RBCs via PKA- and tyrosine kinase p72syk-dependent pathways. ERK signaling is implicated in RBC ICAM-4 phosphorylation, promoting SS RBC adhesion to the endothelium. SS RBC adhesion and phosphorylation of both ERK and ICAM-4 all decreased with continued cell exposure to epinephrine, implying that activation of ICAM-4–mediated SS RBC adhesion is temporally associated with ERK1/2 activation. Furthermore, recombinant ERK2 phosphorylated α- and β-adducins and dematin at the ERK consensus motif. Cytoskeletal protein 4.1 also showed dynamic phosphorylation but not at the ERK consensus motif. These results demonstrate that ERK activation induces phosphorylation of cytoskeletal proteins and the adhesion molecule ICAM-4, promoting SS RBC adhesion to the endothelium. Thus, blocking RBC ERK1/2 activation, such as that promoted by catecholamine stress hormones, could ameliorate SCD pathophysiology.

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Li, Yikun, Xiali Huang, Jingjing Wang, Ruiling Huang, and Dan Wan. "Regulation of Iron Homeostasis and Related Diseases." Mediators of Inflammation 2020 (May 2, 2020): 1–11. http://dx.doi.org/10.1155/2020/6062094.

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The liver is the organ for iron storage and regulation; it senses circulating iron concentrations in the body through the BMP-SMAD pathway and regulates the iron intake from food and erythrocyte recovery into the bloodstream by secreting hepcidin. Under iron deficiency, hypoxia, and hemorrhage, the liver reduces the expression of hepcidin to ensure the erythropoiesis but increases the excretion of hepcidin during infection and inflammation to reduce the usage of iron by pathogens. Excessive iron causes system iron overload; it accumulates in never system and damages neurocyte leading to neurodegenerative diseases such as Parkinson’s syndrome. When some gene mutations affect the perception of iron and iron regulation ability in the liver, then they decrease the expression of hepcidin, causing hereditary diseases such as hereditary hemochromatosis. This review summarizes the source and utilization of iron in the body, the liver regulates systemic iron homeostasis by sensing the circulating iron concentration, and the expression of hepcidin regulated by various signaling pathways, thereby understanding the pathogenesis of iron-related diseases.

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Blue, M. L., D. A. Hafler, K. A. Craig, H. Levine, and S. F. Schlossman. "Phosphorylation of CD4 and CD8 molecules following T cell triggering." Journal of Immunology 139, no. 12 (December 15, 1987): 3949–54. http://dx.doi.org/10.4049/jimmunol.139.12.3949.

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Abstract CD4 and CD8 molecules have been implicated in the regulation of T cell activation. In the present study, CD4 and CD8 were modified by increased phosphorylation when T cell clones or T cells were either exposed to phorbol-12-myristate- 13-acetate or were triggered via the CD3-T cell receptor complex. Activation of T cells through the CD2 sheep erythrocyte binding protein, using anti-T11(2) and -T11(3) antibodies, also resulted in CD4 and CD8 phosphorylation. These findings suggest that signals derived from two different receptor pathways can converge and result in similar molecular modifications of CD4 and CD8. Furthermore, phorbol myristate acetate treatment or activation via the CD2 pathway induced phosphorylation of the CD4 and CD8 molecules of thymocytes, suggesting that these molecules may be functional in thymus. Together, our findings indicate that CD4 and CD8 phosphorylation is a consequence of T cell triggering, and suggest that CD4 and CD8 phosphorylation may represent a molecular signaling mechanism among the CD3-T cell receptor complex, CD2, CD4, and CD8.

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Quinn, Brendan J., and Athar H. Chishti. "Erythrocyte Scaffolding Protein p55 Functions as An Essential Regulator of Neutrophil Polarity." Blood 112, no. 11 (November 16, 2008): 318. http://dx.doi.org/10.1182/blood.v112.11.318.318.

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Abstract Erythrocyte p55 is a prototypical member of a family of scaffolding proteins known as Membrane Associated Guanylate Kinase Homologues (MAGUKs). MAGUKs are multi-domain proteins that couple signals from specialized sites at the plasma membrane to intracellular signal transduction pathways and the cytoskeleton. P55 was originally identified in the erythrocytes as part of a ternary complex with protein 4.1R and glycophorin C, providing a critical linkage between the actin cytoskeleton and the plasma membrane. Although p55 is expressed in a variety of tissues, especially hematopoietic cells, its biological function is unclear. Here, using a p55 knockout mouse model, we show that p55 plays a prominent role in the regulation of neutrophil polarization. Neutrophils are the first respondents during infection and injury, adopting a highly polarized morphology when stimulated with chemotactic factors. G proteincoupled surface receptors recognize the external chemotactic gradient and translate it into an internal gradient of signaling molecules. At the front of the cell, accumulation of the lipid product phosphatidylinositol-3,4,5-trisphosphate (PIP3), activation of the small GTPase Rac, and polymerization of F-actin stimulates a positive feedback loop promoting pseudopod formation. Here, we show that neutrophils lacking p55 form multiple transient pseudopods at the sides and back of the cell upon stimulation. P55 is required for limiting the pseudopod in the direction of chemoattractant. As a result, these neutrophils do not migrate efficiently up a chemotactic gradient in vitro. Biochemical analysis indicates that total F-actin polymerization and total Rac activation is similar between wild type and p55 knockout neutrophils. However, we found that phosphorylation of AKT, the major kinase downstream of the phosphatidylinositol 3-kinase (PI3K)-PIP3 pathway, is almost completely blocked in p55 knockout neutrophils. This finding suggests that p55 exerts its functional effect by regulating PIP3 accumulation or its localization at the membrane, which is responsible for amplification of the frontness signal and stability of the leading edge pseudopod. Consistent with this finding, the p55 null mice are significantly more susceptible to spontaneous and induced infections. Taken together, we have identified p55 as a novel mediator of the frontness signal in neutrophils that promotes polarization and efficient chemotaxis.

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Teixeira, Susana A., Daniele B. D. Marques, Thaís C. Costa, Haniel C. Oliveira, Karine A. Costa, Eula R. Carrara, Walmir da Silva, et al. "Transcription Landscape of the Early Developmental Biology in Pigs." Animals 11, no. 5 (May 18, 2021): 1443. http://dx.doi.org/10.3390/ani11051443.

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Since pre- and postnatal development are programmed during early prenatal life, studies addressing the complete transcriptional landscape during organogenesis are needed. Therefore, we aimed to disentangle differentially expressed (DE) genes between fetuses (at 35 days old) and embryos (at 25 days old) through RNA-sequencing analysis using the pig as model. In total, 1705 genes were DE, including the top DE IBSP, COL6A6, HBE1, HBZ, HBB, and NEUROD6 genes, which are associated with developmental transition from embryos to fetuses, such as ossification, skeletal muscle development, extracellular matrix organization, cardiovascular system, erythrocyte differentiation, and neuronal system. In pathway analysis, embryonic development highlighted those mainly related to morphogenic signaling and cell interactions, which are crucial for transcriptional control during the establishment of the main organs in early prenatal development, while pathways related to myogenesis, neuronal development, and cardiac and striated muscle contraction were enriched for fetal development, according to the greater complexity of organs and body structures at this developmental stage. Our findings provide an exploratory and informative transcriptional landscape of pig organogenesis, which might contribute to further studies addressing specific developmental events in pigs and in other mammals.

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Keykavousi, Keynaz, Fahimeh Nourbakhsh, Nooshin Abdollahpour, Farzaneh Fazeli, Alireza Sedaghat, Vahid Soheili, and Amirhossein Sahebkar. "A Review of Routine Laboratory Biomarkers for the Detection of Severe COVID-19 Disease." International Journal of Analytical Chemistry 2022 (October 11, 2022): 1–14. http://dx.doi.org/10.1155/2022/9006487.

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As the COVID-19 pandemic continues, there is an urgent need to identify clinical and laboratory predictors of disease severity and prognosis. Once the coronavirus enters the cell, it triggers additional events via different signaling pathways. Cellular and molecular deregulation evoked by coronavirus infection can manifest as changes in laboratory findings. Understanding the relationship between laboratory biomarkers and COVID-19 outcomes would help in developing a risk-stratified approach to the treatment of patients with this disease. The purpose of this review is to investigate the role of hematological (white blood cell (WBC), lymphocyte, and neutrophil count, neutrophil-to-lymphocyte ratio (NLR), platelet, and red blood cell (RBC) count), inflammatory (C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), and lactate dehydrogenase (LDH)), and biochemical (Albumin, aspartate aminotransferase (AST) and alanine aminotransferase (ALT), blood urea nitrogen (BUN), creatinine, D-dimer, total Cholesterol, low-density lipoprotein (LDL), and high-density lipoprotein (HDL)) biomarkers in the pathogenesis of COVID-19 disease and how their levels vary according to disease severity.

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Van Vuren, Annelies J., Stephanie van Straaten, Michal Mokry, Richard van Wijk, and Eduard J. van Beers. "A Unique Monocyte Transcriptome Discriminates Sickle Cell Disease from Other Hereditary Hemolytic Anemias and Shows the Particular Importance of Lipid and Interferon Signaling." Blood 134, Supplement_1 (November 13, 2019): 980. http://dx.doi.org/10.1182/blood-2019-124070.

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Introduction Sickle cell disease (SCD) is a hereditary hemolytic disease characterized by a complex pathophysiology including inflammation and oxidative stress. Intravascular hemolysis leads to release of heme, an erythrocyte-derived Danger Associated Molecular Pattern (e-DAMP) that augments Toll Like Receptor 4 (TLR4) signaling. TLR4 signaling is important in development of acute and chronic complications in SCD. We investigated gene expression profiles of TLR4+ cells (by positive selection of its co-receptor CD14) of patients with SCD and other hemolytic anemias to identify differential regulated pathophysiological pathways. Methods Analyses were performed in 60 adults with hereditary hemolytic anemia and 10 healthy controls included in the ZEbRA cohort (UMC Utrecht, [NL5189]). Patients treated with systemic anti-inflammatory drugs were excluded (n=1). Deferasirox (DFX)-treated SCD patients were analyzed separately as DFX ameliorated pro-inflammatory effects of heme. CD14+ cells were isolated using anti-CD14 microbeads. RNA sequencing was performed on a Nextseq500 platform (Illumina) using a single-end 75bp high-output run. Differentially expressed genes (DEGs) were identified using DESeq2 v1.24.0 in R. To correct for presence of reticulocytes and lymphocytes, data were corrected for expression of 4 hemoglobin (HBA1, HBB, HBG1, HBG2) and 4 T cell specific genes (CD3E, CD3D, CD3G, CD247). A list was constructed from all genes differentially expressed (adj. p<0.01; absolute log2fold change >1) in both the comparisons of SCD without DFX versus other hemolytic anemias and of SCD without DFX versus healthy controls. Pathway enrichment analysis (ReactomePA v1.28.0 in R) was performed with preselected DEGs (adj. p<0.1; absolute log2fold change >0.5). P-values were adjusted with the Benjamini-Hochberg procedure. Results Demographics and hematological values are provided in Table 1. Principal component analysis based on RNA sequencing data separated SCD patients without DFX from hemolytic anemia patients, healthy controls and SCD patients with DFX (Figure 1). Analysis of DEGs discriminating SCD patients from both patients with other hemolytic anemias and healthy controls rendered 29 genes (Figure 2). Heme oxygenase-1 (HMOX1) was one of these genes (versus healthy controls adj. p=5.6E-13; versus other hemolytic anemias adj. p=3.3E-15) and this is in line with the hypothesis that intravascular free heme is an important effector of gene regulation in monocytes. This sets SCD apart from the other studied hemolytic anemias. The other 28 genes included, PPARG, GUCY1A1, KLF5 and CXCR3 signaling (CXCL9 and CXCL11) which are associated with vascular remodeling and development of pulmonary hypertension. The list of 29 DEGs highlights interesting differences in gene expression of two processes related to immune signaling: CXCR3 signaling by CXCL9 and CXCL11, and lipid metabolism (STARD4, DLC1, SQLE, ME1). Pathway enrichment analysis showed enrichment of genes involved in IFN signaling (type I and II) in SCD versus healthy controls (adj. p=4.4E-16). And, in line with the list of 29 genes, enrichment of genes concerning chemokine signaling in SCD versus both healthy controls and other hemolytic anemias (respectively adj. p=0.06 and adj. p=0.01) and cholesterol biosynthesis in SCD versus other hemolytic anemias (adj. p=0.09). Conclusion Our data shows the unique inflammatory profile of SCD monocytes as opposed to other hemolytic anemias. Moreover, it suggests that lipid metabolism and IFN signaling are important differentiating immune signaling pathways. It is known that alterations in plasma lipid levels in SCD relate to hemolytic severity and vasculopathy. Our data suggests an important role for lipid biology in SCD monocytes. We hypothesize on an important contribution of cholesterol accumulation in enhancement of TLR4 signaling, as lipid rafts accelerated Nf-kB activation in macrophages. (Lee et al., Nat. Commun. 2017; Koseki et al., J. Lip. Res. 2007) The importance of type I and II IFN signaling in SCD suggests widespread involvement of the immune system. IFNg-inducible cytokines CXCL9 and CXCL11 are associated with Th1 polarization and activation. In summary, the data support the unique role of monocyte immune signaling in SCD. Furthermore, we identified pathways that seem to be relevant for immune regulation and thereby for development of disease complications. Disclosures van Wijk: Agios Pharmaceuticals: Consultancy, Research Funding; RR Mechatronics: Research Funding. van Beers:Agios Pharmaceuticals, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Research Funding; RR Mechatronics: Research Funding; Novartis: Consultancy, Research Funding.

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Gou, Panhong, Wenchao Zhang, and Stephane Giraudier. "Insights into the Potential Mechanisms of JAK2V617F Somatic Mutation Contributing Distinct Phenotypes in Myeloproliferative Neoplasms." International Journal of Molecular Sciences 23, no. 3 (January 18, 2022): 1013. http://dx.doi.org/10.3390/ijms23031013.

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Myeloproliferative neoplasms (MPN) are a group of blood cancers in which the bone marrow (BM) produces an overabundance of erythrocyte, white blood cells, or platelets. Philadelphia chromosome-negative MPN has three subtypes, including polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). The over proliferation of blood cells is often associated with somatic mutations, such as JAK2, CALR, and MPL. JAK2V617F is present in 95% of PV and 50–60% of ET and PMF. Based on current molecular dynamics simulations of full JAK2 and the crystal structure of individual domains, it suggests that JAK2 maintains basal activity through self-inhibition, whereas other domains and linkers directly/indirectly enhance this self-inhibited state. Nevertheless, the JAK2V617F mutation is not the only determinant of MPN phenotype, as many normal individuals carry the JAK2V617F mutation without a disease phenotype. Here we review the major MPN phenotypes, JAK-STAT pathways, and mechanisms of development based on structural biology, while also describing the impact of other contributing factors such as gene mutation allele burden, JAK-STAT-related signaling pathways, epigenetic modifications, immune responses, and lifestyle on different MPN phenotypes. The cross-linking of these elements constitutes a complex network of interactions and generates differences in individual and cellular contexts that determine the phenotypic development of MPN.

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Potti, Anil, Holly K. Dressman, and Murat O. Arcasoy. "Gene Expression Patterns Identify Novel Biologically Relevant Signaling and Transcriptional Pathways Involved in Terminal Erythroid Differentiation and Polycythemia Vera." Blood 106, no. 11 (November 16, 2005): 3524. http://dx.doi.org/10.1182/blood.v106.11.3524.3524.

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Abstract Hematopoietic proliferation, lineage commitment, and terminal differentiation are characterized by the emergence of a cell type-specific gene expression and transcriptional programs that determine the specific phenotype and function of cells in the erythroid lineage. Our objectives in this study were to identify unique gene expression patterns that characterize the transcriptional program of normal primary human erythroid precursors during terminal differentiation, and define the gene expression patterns seen in erythroblasts (EBL) of patients with polycythemia vera (PV). Homogenous populations of primary proEBL were generated from purified liquid cultures of CD34+ cells collected from healthy volunteers and PV patients. All patients with PV were diagnosed based on established criteria and had the JAK2-V617F mutation. Morphologic examination and surface expression of CD71 confirmed the purity of proEBL cell populations. ProEBL from normal individuals were induced to terminally differentiate generating orthochromatic EBL. RNA was extracted from normal proEBL, PV proEBL, and normal orthochromatic EBL. Affymetrix U133 Plus 2.0 arrays representing approximately 39,000 human genes were used for gene expression analysis. Four replicates from four independent primary cell cultures were analyzed for each comparison group (e.g. undifferentiated proEBL versus terminally differentiated orthochromatic EBL). Unsupervised hierarchical clustering showed distinct gene expression profiles in the proEBL and terminally differentiated EBL lineages. 1109 genes (2.0 fold change, P<0.01) were found to be differentially expressed. Numerous erythroid genes were found to be upregulated during terminal differentiation [e.g. globin genes, erythropoietin receptor, heme synthesis enzymes (ferrochelatase, ALAS2) erythrocyte membrane proteins (band 3, ankyrin, protein 4.1) and transcription factors (NFE2, Kruppel-like factors, myb, GATA2)]. As a proof of validation, the differential expression of 7 genes was verified by Northern blotting. To better understand the biologic role of the gene sets identified, using Ingenuity pathway analysis, individual genes were integrated into specific regulatory and signaling pathway networks. A total of 19 networks with significant scores (>23) were identified. Biological functions of the identified networks included RNA post-transcriptional regulation, cell cycle control, translational regulation, DNA replication and repair and cellular assembly/organization. In a proof of principle study, gene expression patterns in PV proEBL (n=6) were compared to normal proEBL (n=5). Unsupervised hierarchical clustering showed a distinct gene expression profile for PV. A binary regression predictive model was also developed to find gene expression patterns predictive for PV. Using this model a 150 gene predictor was found that could predict PV patients from control at 100% accuracy. Ingenuity pathways analysis of a subset of gene subsets demonstrated several biologically relevant networks that were distinct in patients with PV, including myc, CDC2, and JAK2. Deregulation of normal transcriptional mechanisms in hematopoietic cells is associated with the pathogenesis of PV. Further, our data shows that genomic studies provide new insights into transcriptional programs that govern erythroid differentiation, and identify biologically relevant deregulated pathways as potential targets for therapy in PV.

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Song, Anren, Cheng Zhao, Yujin Zhang, Jessica Li, Michael R. Blackburn, Almut Grenz, Holger Eltzschig, Rodney E. Kellems, and Yang Xia. "Functions and Regulation of Erythrocyte Equlibrative Nucleoside Transporter 1 (ENT1) in Acute Hypoxia Mediated Tissue Injury." Blood 124, no. 21 (December 6, 2014): 2666. http://dx.doi.org/10.1182/blood.v124.21.2666.2666.

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Abstract Using a nonbiased high throughput metabolomic screen, coupled with genetic and pharmacological approaches, recent studies demonstrated that excessive adenosine signaling through the A2B adenosine receptor triggers sickling by induction of 2,3-bisphosphoglycerate (2,3-BPG), an erythroid specific metabolite that induces O2 release from hemoglobin. Adenosine is a signaling nucleoside that elicits numerous physiological and pathological effects by engaging membrane receptors. Notably, equlibrative nucleoside transporters (ENTs) on erythrocytes have been long speculated to regulate extracellular adenosine concentrations under hypoxic conditions. Thus, we hypothesize that ENT is likely a key molecule responsible for elevated circulating adenosine levels and protects tissues from hypoxia induced injury. To test this hypothesis, we first conducted in vivo Carbon-14 labeled adenosine (C14-Ado) injection and in vitro functional C14-Ado uptake assays. We found that erythrocyte plays a key role in regulation of circulating adenosine. We then conducted western blot analysis to compare expression profiles of ENTs on erythrocyte. We found that ENT1 is the major ENT expressed on both mouse and human erythrocytes. Using genetic approach, we successfully generated an erythrocyte ENT1 knockout mouse model. Using this genetic model and pharmacological approach combined with in vivo C14-Ado injection and in vitro C14-Ado uptake assay, we demonstrated that ENT1 1) is the major adenosine transporter in erythrocyte and 2) erythrocyte is the major cell type involved in regulating circulating adenosine levels through ENT1’s function. Using erythrocyte ENT1 knockout mouse model, we found that, during acute hypoxia treatment, the loss of erythrocyte ENT1 can cause faster increase in circulating adenosine level, subsequently promoting 2,3-BPG production, triggering oxygen release, and protecting acute hypoxia-mediated tissue injury. Mechanistically, we demonstrated that hypoxia regulates ENT1 activity through adenosine-ADORA2B-PKA signaling pathway. Overall, our studies demonstrate that 1) ENT1 is a major adenosine transporter expressed by erythrocytes and erythrocytes are the major cell type responsible for regulating circulating adenosine. 2) Hypoxia regulates ENT1 activity through adenosine-ADORA2B-PKA signaling pathway. 3) Inhibition or deletion of erythrocyte ENT1 results in enhanced adenosine-mediated 2,3-BPG induction and hemoglobin deoxygenation in RBCs when hypoxia is encountered. Thus, our findings suggest that erythrocyte ENT1 and ADORA2B are novel targets to prevent hypoxia-mediated tissue injury. Disclosures No relevant conflicts of interest to declare.

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Mohseni, Morvarid, Anwar Khan, and Athar H. Chishti. "Erythrocyte Dematin Regulates Glucose Transport Via Akt Phosphorylation and 14-3-3zeta Association." Blood 114, no. 22 (November 20, 2009): 1990. http://dx.doi.org/10.1182/blood.v114.22.1990.1990.

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Abstract Abstract 1990 Poster Board I-1012 Erythrocyte dematin is a widely expressed actin-binding and bundling protein, and functions as a suppressor of RhoA signaling in fibroblasts (Mohseni and Chishti, Molecular Cell Biology 28: 4712-4718, 2008). Dematin is a substrate of multiple protein kinases, and its actin bundling activity is regulated by cAMP dependent protein kinase. Recently, we identified a novel interaction between dematin and glucose transporter-1 (GLUT1) that is critically important for erythrocyte shape and membrane mechanical properties (Khan et al., Journal of Biological Chemistry 283:14600-14609, 2008). Since homologues of dematin and GLUT1 exist in many non-erythroid cells, we proposed that a conserved mechanism might couple related sugar transporters, such as the insulin-responsive glucose transporter-4 (GLUT4), to the actin cytoskeleton via dematin. Immunocytochemistry established the presence of dematin in 3T3-L1 adipocytes, and a small pool of dematin and GLUT4-containing vesicles co-localized in 3T3-L1 cells under both basal and insulin-stimulated conditions. Plasma membrane sheet assays indicate that upon insulin stimulation, dematin translocates to the plasma membrane along with GLUT4, resulting in partial co-localization at the plasma membrane. Furthermore, dematin RNAi treated 3T3-L1 cells show reduced GLUT4 protein expression, suggesting that dematin may regulate a sub-population of GLUT4 via the lysosomal degradation pathway in adipocytes. Importantly, glucose transport was reduced by ∼28% in 3T3-L1 adipocytes depleted of dematin, and by ∼15% in the dematin headpiece knockout (HPKO) mouse primary adipocytes. Since a significant amount of dematin did not co-localize with GLUT4 in the cytosol and plasma membrane, biochemical interaction between dematin and GLUT4 could not be verified using immunoprecipitation and transfection assays. Although dematin does not bind directly to GLUT4 under these conditions, a possibility existed that this interaction may be transient and mediated through an adaptor protein. Interestingly, dematin contains seven 14-3-3 binding sites, and 14-3-3 adaptor has been shown to be functionally involved in GLUT4 trafficking. We demonstrate that phosphorylated dematin binds to 14-3-3 in 3T3-L1 adipocytes under both basal and insulin stimulated conditions. Mutagenesis studies identify serine-85 on dematin as the primary phospho-binding site for 14-3-3zeta. Furthermore, using pharmacological inhibitors, Akt is identified as the likely protein kinase that phosphorylates dematin to mediate the biochemical interactions between dematin and 14-3-3zeta. Together, our results identify erythrocyte dematin as a potential regulator of glucose transporter trafficking and degradation pathways in adipocytes with functional implications for glucose homeostasis, diabetes, and obesity. Disclosures: No relevant conflicts of interest to declare.

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Cifani, Paolo, Mojdeh Shakiba, Fiona Brown, Avantika Dhabaria, Daniel E. Bauer, and Alex Kentsis. "Functional Proteomics of Regulatory and Epigenetic Signaling in Normal and Malignant Hematopoiesis." Blood 126, no. 23 (December 3, 2015): 4972. http://dx.doi.org/10.1182/blood.v126.23.4972.4972.

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Abstract Post-translational modifications of proteins are increasingly recognized as key regulators of functional complexes assembly and cellular enzymes activity, processes that control both normal and aberrant cell growth and development. To enable comprehensive, sensitive and quantitative analysis of these processes, we adapted recently developed high-efficiency nanoscale multidimensional liquid chromatography with high-resolution Orbitrap mass spectrometry. Termed accumulated ion monitoring (AIM), this mass spectrometry method achieved nearly 7 orders of magnitude of quantitative accuracy and absolute limit of detection of 600 molecules per scan, enabling the study of rare cell populations (Fig. 1). We leveraged AIM mass spectrometry to develop a panel of 1583 synthetic reference peptides, based on global and published proteomics maps of normal human and leukemia cells. This Quantitative Cell Proteomics Atlas (QCPA) profiles 384 key effectors of cell surface signaling, proliferation, quiescence, stress response, and epigenetic control of gene expression (Fig. 2). QCPA enables unprecedented accuracy and sensitivity for the functional analysis of rare cells, with focus on protein regulation via phosphorylation, acetylation and methylation, as well as on concentration (http://alexkentsis.net/qcpa). By using functional proteomics profiling of primary human CD34+ and acute myeloid leukemia (AML) cells, we identify new pathways controlling erythrocyte differentiation and AML therapy resistance, as facilitated by a newly developed program ProteoModlR (http://github.com/kentsisresearchgroup/ProteoModlR). AIM mass spectrometry and QCPA functional proteomics are rapidly adaptable and generalizable tools for the investigation of regulatory and epigenetic signaling in normal and diseased cells. Figure 1. Limits of detection and quantitation for a serially diluted synthetic peptide from human Myocyte-specific Enhancer Factor 2C. Figure 1. Limits of detection and quantitation for a serially diluted synthetic peptide from human Myocyte-specific Enhancer Factor 2C. Figure 2. Functional classification of proteins included in the QCPA. Figure 2. Functional classification of proteins included in the QCPA. Disclosures Bauer: Editas Medicine: Consultancy; Biogen: Research Funding.

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Peng, Zhangzhe, Renna Luo, Tingting Xie, Weiru Zhang, Hong Liu, Wei Wang, Lijian Tao, Rodney E. Kellems, and Yang Xia. "Erythrocyte Adenosine A2B Receptor-Mediated AMPK Activation: A Missing Component Counteracting CKD by Promoting Oxygen Delivery." Journal of the American Society of Nephrology 30, no. 8 (July 5, 2019): 1413–24. http://dx.doi.org/10.1681/asn.2018080862.

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BackgroundOxygen deprivation or hypoxia in the kidney drives CKD and contributes to end organ damage. The erythrocyte’s role in delivery of oxygen (O2) is regulated by hypoxia, but the effects of CKD are unknown.MethodsWe screened all of the metabolites in the whole blood of mice infused with angiotensin II (Ang II) at 140 ng/kg per minute up to 14 days to simulate CKD and compared their metabolites with those from untreated mice. Mice lacking a receptor on their erythrocytes called ADORA2B, which increases O2 delivery, and patients with CKD were studied to assess the role of ADORA2B-mediated O2 delivery in CKD.ResultsUntargeted metabolomics showed increased production of 2,3-biphosphoglycerate (2,3-BPG), an erythrocyte-specific metabolite promoting O2 delivery, in mice given Ang II to induce CKD. Genetic studies in mice revealed that erythrocyte ADORA2B signaling leads to AMPK-stimulated activation of BPG mutase, promoting 2,3-BPG production and O2 delivery to counteract kidney hypoxia, tissue damage, and disease progression in Ang II–induced CKD. Enhancing AMPK activation in mice offset kidney hypoxia by triggering 2,3-BPG production and O2 delivery. Patients with CKD had higher 2,3-BPG levels, AMPK activity, and O2 delivery in their erythrocytes compared with controls. Changes were proportional to disease severity, suggesting a protective effect.ConclusionsMouse and human evidence reveals that ADORA2B-AMPK signaling cascade–induced 2,3-BPG production promotes O2 delivery by erythrocytes to counteract kidney hypoxia and progression of CKD. These findings pave a way to novel therapeutic avenues in CKD targeting this pathway.

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Prykhozhij, Sergey, Lucia Caceres, Kevin Ban, Christopher McMaster, Johane M. Robitaille, and Jason N. Berman. "Loss of iqgap1 (IQ Motif Containing GTPase Activating Protein 1) in Zebrafish Leads to Intracerebellar Hemorrhage, Morphological Abnormalities and Activates Hematopoietic Response." Blood 138, Supplement 1 (November 5, 2021): 2071. http://dx.doi.org/10.1182/blood-2021-148029.

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Abstract Defects in multiple cell signaling molecules lead to disruptions of vascular integrity given the need for fine-tuned regulation of the cell adhesion complexes. These genetic defects have been linked to the development of intracerebral hemorrhage (ICH). There is genetic evidence in humans for ICH due to some genetic variants, while other variants have been identified in preclinical animal models. Signaling adaptor proteins play a crucial role in cell signaling by promoting interactions between effector proteins and even enabling integration of different pathways. IQGAP1 is a conserved signaling adaptor known for its roles in cell adhesion, cancer and for other cell biological effects. We engineered a zebrafish null mutant in the zebrafish iqgap1 gene by introducing an 11-bp deletion using a CRISPR/Cas9 genome editing method and characterized its phenotype. Homozygous mutants exhibit severe brain hemorrhage and morphological abnormalities, which are ultimately lethal, in about 30-40% of cases, whereas the other embryos survive to adulthood. We visualized the expression pattern of iqgap1 relative to the established fli1a vascular marker and found that iqgap1 strongly overlapped with fli1a expression, but was expressed much more broadly in tissues, such as muscle, branchial arches, and the caudal hematopoietic tissue (equivalent to the mammalian fetal liver). Critically, iqgap1 exhibited co-localization with fli1a in the blood vessels of the central nervous system, whose disruption is likely responsible for the brain hemorrhage. Whole embryo RNA sequencing-based comparison of hemorrhage-positive iqgap1-/- embryo pools with wild-type embryos at 52 hours post-fertilization (hpf) shortly after the onset of hemorrhage identified approximately 800 differentially regulated genes. The most striking feature of this dataset was up-regulation of hematopoietic markers especially those of erythrocytes, neutrophils, mast cells and HSPCs (hematopoietic stem and progenitor cells), but not macrophages. We have confirmed by in situ hybridization with marker gene probes that erythrocyte and neutrophil production is up-regulated most strongly in iqgap1-/- embryos undergoing some level of hemorrhage. By contrast, fli1a endothelial and stem cell marker was downregulated. This animal model provides a compelling genotype-phenotype correlation, implicating IQGAP1 as a new player in vascular disorders such as ICH and identifying a previously unrecognized relationship between IQGAP and regulation of hematopoiesis. Furthermore, this model is now poised to identify ameliorating and exacerbating modifier lesions and potential therapeutic agents that restore normal vascular integrity and prevent ICH. Disclosures Robitaille: Novartis: Consultancy. Berman: Oxford Immune Algorithmics: Membership on an entity's Board of Directors or advisory committees.

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Wang, Yi, Zhihua Zhou, Li Chen, Xiangwei He, Hui Li, Yingru Huang, and Yu Pu. "Efficacy of Duhuo Jisheng Decoction in Treating Ankylosing Spondylitis: Clinical Evidence and Potential Mechanisms." Evidence-Based Complementary and Alternative Medicine 2022 (April 4, 2022): 1–12. http://dx.doi.org/10.1155/2022/3305773.

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Background. Duhuo Jisheng Decoction (DHJSD) is an ancient compound widely used in the treatment of ankylosing spondylitis (AS). However, its efficacy is controversial, and its mechanism of action is not clear enough. Using meta-analysis and network pharmacology, our study evaluated the clinical efficacy of DHJSD in the treatment of AS and explored its mechanisms of action. Methods. We searched medical databases, including Embase, PubMed, the China National Knowledge Infrastructure databases, Wanfang, and the Chinese Scientific Journal Database, to identify studies that met the inclusion criteria. RevMan 5.3 software was used for the meta-analysis. The compounds and the potential protein targets of DHJSD were obtained from the Traditional Chinese Medicine Systems Pharmacology (TCMSP) database and analysis platform. AS was treated as a search query in the NCBI, PharmGKB, TTD, DrugBank, and OMIM databases to obtain disease-related genes. The overlapping targets of DHJSD and AS were identified, and then Gene Ontology functional enrichment and Kyoto Encyclopedia of Genes and Genomes enrichment analyses were performed. Cytoscape was employed to construct a drug-compound-target network and a protein-protein interaction (PPI) network. CytoHubba was utilized to select the hub genes. Results. A total of 10 studies involving 860 participants were included in the meta-analysis. Compared with the control, DHJSD treatment significantly improved clinical symptoms; reduced the erythrocyte sedimentation rate (ESR), the C-reactive protein (CPR), and interleukin 6 (IL-6) levels; increased the degree of motion of the chest; reduced the visual analog scale (VAS) pain score; reduced Schober’s test values; reduced the finger-to-floor distance; reduced the duration of morning stiffness. However, the differences were not statistically significant in the Bath Ankylosing Spondylitis Functional Index scores, the Bath Ankylosing Spondylitis Disease Activity Index scores, the bone Gla-containing protein (BGP) levels, or the bone alkaline phosphatase (BALP) levels. In terms of adverse events, DHJSD treatment of AS reduced the incidence of gastrointestinal events, the incidence of skin events, and the incidence of abnormal liver function; however, there was no statistically significant reduction in the incidence of adverse renal function events. Subgroup analysis showed that in the treatment of AS, the clinical effect of DHJSD for AS was better than that of the controls for both treatment durations, ≤2 months and >2 months. A total of 178 active compounds and 47 related potential targets were identified for DHJSD in the treatment of AS, including four hub genes (CXCL8, PTGS2, VEGFA, and STAT3). The core active ingredients of DHJSD in the treatment of AS were mainly quercetin, kaempferol, licochalcone A, and isorhamnetin. DHJSD treatment of AS-related pathways mainly involved the IL-17 signaling pathway, the TNF signaling pathway, and the rheumatoid arthritis signaling pathway. Conclusion. The above results suggest that DHJSD acts on AS through multiple targets, components, and pathways with significant clinical efficacy. Future studies may further explore the active components of DHJSD.

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Wara, Akm Khyrul, Kevin Croce, ShiYin Foo, Xinghui Sun, Basak Icli, Yevgenia Tesmenitsky, Zhuoxiao Cao, et al. "Kruppel-Like Factor 10 (KLF10)-Deficient Mice Have Marked Defects In EPC Differentiation, Function, and Angiogenesis." Blood 116, no. 21 (November 19, 2010): 4314. http://dx.doi.org/10.1182/blood.v116.21.4314.4314.

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Abstract Abstract 4314 Background: Emerging evidence demonstrates that endothelial progenitor cells (EPCs) may originate from the bone marrow and are capable of being recruited to sites of ischemic injury and contribute to neovascularization. However, the identities of these bone marrow cells and the signaling pathways that regulate their differentiation into functional EPCs remain poorly understood. Methods and Results: We previously identified that among hematopoietic progenitor stem cells, common myeloid progenitors (CMPs) and granulocyte-macrophage progenitors (GMPs) can preferentially differentiate into EPCs and possess high angiogenic activity under ischemic conditions compared to megakaryocyte-erythrocyte progenitors (MEPs), hematopoietic stem cells (HSCs), and common lymphoid progenitors (CLPs). Herein, we identify that a TGF-β1-responsive Kruppel-like Factor, KLF10, is robustly expressed in EPCs derived from CMPs and GMPs, compared to progenitors lacking EPC markers. KLF10–/– mice have marked defects in circulating EPCs (–23.6% vs. WT, P<0.004). In addition, EPC differentiation and TGF-β induced KDR responsiveness is markedly impaired (CMPs: WT 22.3% vs. KO 8.64%, P<0.0001; GMPs: WT 32.8% vs. KO 8.97%, P<0.00001). Functionally, KLF10–/– EPCs derived from CMPs and GMPs adhered less to fibronectin-coated plates (CMPs: WT 285 vs. KO 144.25, P< 0.0004; GMPs: WT 275.25 vs. KO 108.75, P <0.0003) and had decreased rates of migration in transwell Boyden chambers (CMPs: WT 692 vs. KO 298.66, P<0.00004; GMPs: WT 635.66 vs. KO 263.66, P<0.00001). KLF10–/– mice displayed impaired blood flow recovery after hindlimb ischemia (day 14, WT 0.827 vs. KO 0.640, P <0.009), an effect completely rescued by WT EPCs, but not KLF10–/– EPCs. Matrigel plug implantation studies demonstrated impaired angiogenesis in KLF10–/– mice compared to WT mice (WT 158 vs. KO 39.83, P<0.00000004). Overexpression studies revealed that KLF10 rescued EPC formation from TGF-β1+/– CMPs and GMPs. Mechanistically, TGF-β1 and KLF10 target the VEGFR2 promoter in EPCs which may underlie these effects. Background: Collectively, these observations identify that TGF-β1 signaling and KLF10 are part of a key signaling pathway that regulates EPC differentiation from CMPs and GMPs and may provide a therapeutic target during cardiovascular ischemic states. Disclosures: No relevant conflicts of interest to declare.

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Zhang, Jian, Chi Huang, Zehan Liu, Shuai Ren, Zilong Shen, Kecheng Han, Weiguang Xin, Guanyi He, and Jianyu Liu. "Screening of Potential Biomarkers in the Peripheral Serum for Steroid-Induced Osteonecrosis of the Femoral Head Based on WGCNA and Machine Learning Algorithms." Disease Markers 2022 (February 10, 2022): 1–17. http://dx.doi.org/10.1155/2022/2639470.

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Background. Steroid-induced osteonecrosis of the femoral head (SONFH) has produced a substantial burden of medical and social experience. However, the current diagnosis is still limited. Thus, this study is aimed at identifying potential biomarkers in the peripheral serum of patients with SONFH. Methods. The expression profile data of SONFH (number: GSE123568) was acquired from the Gene Expression Omnibus (GEO) database. Differentially expressed genes (DEGs) in SONFH were identified and used for weighted gene coexpression network analysis (WGCNA). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed to investigate the biological functions. The protein-protein interaction (PPI) network and machine learning algorithms were employed to screen for potential biomarkers. Gene set enrichment analysis (GSEA), transcription factor (TF) enrichment analysis, and competing endogenous RNA (ceRNA) network were used to determine the biological functions and regulatory mechanisms of the potential biomarkers. Results. A total of 562 DEGs, including 318 upregulated and 244 downregulated genes, were identified between SONFH and control samples, and 94 target genes were screened based on WGCNA. Moreover, biological function analysis suggested that target genes were mainly involved in erythrocyte differentiation, homeostasis and development, and myeloid cell homeostasis and development. Furthermore, GYPA, TMCC2, and BPGM were identified as potential biomarkers in the peripheral serum of patients with SONFH based on machine learning algorithms and showed good diagnostic values. GSEA revealed that GYPA, TMCC2, and BPGM were mainly involved in immune-related biological processes (BPs) and signaling pathways. Finally, we found that GYPA might be regulated by hsa-miR-3137 and that BPGM might be regulated by hsa-miR-340-3p. Conclusion. GYPA, TMCC2, and BPGM are potential biomarkers in the peripheral serum of patients with SONFH and might affect SONFH by regulating erythrocytes and immunity.

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da Palma Guerreiro, Alexandra, Cornelia Dorweiler, Ismini Halmer, Olaf Merkel, Elena Maria Hartmann, Valeska Berg, Nina Reinart, et al. "FcmR Shapes BCR Signaling in IgM-Positive Leukemia." Blood 132, Supplement 1 (November 29, 2018): 2620. http://dx.doi.org/10.1182/blood-2018-99-118352.

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Abstract Background: The Fc receptor for IgM (FcmR/ TOSO) is significantly overexpressed on chronic lymphocytic leukemia (CLL) cells from peripheral blood, but becomes down-regulated in the tumor microenvironment by e.g. CD40:CD40L interaction. Since the functional role of FcmR on lymphomagenesis is still not understood, we developed a conditional knockout mouse with B cell-specific FcmR-depletion. These mice were crossbred with the Eµ-TCL1 murine model, which develops a CLL-like phenotype. Results: The depletion of FcmR/TOSO in TCL1 mice (Eµ-Tcl1tg/wt FcmRfl/fl CD19cre/wt; further on called TCT) revealed a significantly shorter overall survival (296 days; n=40) compared to the TOSO expressing control mice (Eµ-Tcl1tg/wt FcmRwt/wt CD19cre/wt; TC; 344 days; n=106; Log-rank p<0.0001). In addition, these mice show a significantly higher blood leukocyte count and lower platelet and erythrocyte count. Leukocytes could be identified as CLL-characteristic leukemic CD19+/CD5+ B cells. Altogether TCT exhibited a faster progress of disease. Spleen immunohistochemistry revealed the transformation of most TCT (14/17 transformed) into an even more aggressive phenotype with increased splenomegaly and change in tissue and cell morphology compared to TC (9/9 not transformed). While characterizing these cells by flow cytometry, we identified a significantly higher expression of IgM on malignant B cells from TCT in comparison to TC mice. This finding indicates that the BCR itself might have a different contribution to lymphomagenesis in FcmR knock-out settings. Therefore, to validate the functional role of FcmR in the process of lymphomagenesis, we performed transcriptome profiling by RNA-Seq using splenic leukemic cells (CD19+ CD5+) from 36-week old TC (n=4) and TCT (n=4) mice. 2089 genes were found to be significantly modulated in the malignant cells of TCT mice, from which 1221 were downregulated and 868 showed an upregulation (significant change in mean expression; p<0.05). To investigate the role of IgM on TCT mice, purified malignant B cells were incubated for two hours with F(ab')2 goat anti-mouse IgM. Strikingly, TCT mice showed 3941 genes (2054 downregulated, 1887 upregulated) with significant difference in expression compared to TC (p<0.05). The gene expression profiles of the anti-IgM treated mice revealed a stronger regulation of BCR signalling in TCT mice, suggesting that FcmR represents an important factor in these processes. We examined the gene expression profiles, using Ingenuity Pathway Analysis Software. Analysis revealed that the most deregulated functions include interferon-signalling, recruitment of leukocytes, infection of cells and cellular movement. Conclusion: Here we present functional evidence that loss of FcmR results in increased IgM/BCR on the surface of non-switched leukemia. Moreover, malignant cells with loss of FcmR are more susceptible to BCR stimulation and show a signature of signalling pathways, which contribute to inflammation in B cell malignancies. Disclosures Fingerle-Rowson: MorphoSys: Employment. Pallasch:Gilead: Research Funding. Wendtner:Abbvie: Consultancy, Honoraria, Other: travel support, Research Funding; Mundipharma: Consultancy, Honoraria, Research Funding; Gilead: Consultancy, Honoraria, Research Funding; GlaxoSmithKline: Consultancy, Honoraria, Other: travel support, Research Funding; Gilead: Consultancy, Honoraria, Other: travel support, Research Funding; Genetech: Consultancy, Honoraria, Other: travel support, Research Funding; Janssen: Consultancy, Honoraria, Other: travel support, Research Funding; Pharmacyclics: Consultancy, Honoraria, Other: travel support, Research Funding; MorphoSys: Consultancy, Honoraria, Other: travel support, Research Funding; Roche: Consultancy, Honoraria, Other: travel support, Research Funding.

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Thuet, K. M., E. A. Bowles, M. L. Ellsworth, R. S. Sprague, and A. H. Stephenson. "The Rho kinase inhibitor Y-27632 increases erythrocyte deformability and low oxygen tension-induced ATP release." American Journal of Physiology-Heart and Circulatory Physiology 301, no. 5 (November 2011): H1891—H1896. http://dx.doi.org/10.1152/ajpheart.00603.2011.

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Low oxygen (O2) tension and mechanical deformation are stimuli for ATP release from erythrocytes. It has been shown previously that rabbit erythrocytes made less deformable with diamide, a thiol cross-linking agent, release less ATP in response to low O2 tension, suggesting a link between these two stimuli. In nonerythroid cells, activation of the Rho/Rho kinase signaling pathway has been reported to decrease cell deformability by altering Rho kinase-dependent cytoskeleton-protein interactions. We investigated the hypothesis that the Rho kinase inhibitor Y-27632 would increase erythrocyte deformability and thereby increase low O2 tension-induced ATP release from erythrocytes. Here we show that Y-27632 (1 μM) increases erythrocyte deformability (5%) and increases low O2 tension-induced ATP release (203%) from healthy human erythrocytes. In addition, we found that, when erythrocytes were made less deformable by incubation with diamide (100 μM), Y-27632 restored both deformability and low O2 tension-induced ATP release to levels similar to those measured in the absence of diamide. These findings suggest that the Rho kinase inhibitor Y-27632 is able to reverse the diamide-induced decrease in erythrocyte deformability and rescue low O2 tension-induced ATP release. These results further support a link between erythrocyte deformability and ATP release in response to low O2 tension.

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Misiti, Francesco, Federica Orsini, M. Elisabetta Clementi, Daniele Masala, Ester Tellone, Antonio Galtieri, and Bruno Giardina. "Amyloid peptide inhibits ATP release from human erythrocytes." Biochemistry and Cell Biology 86, no. 6 (December 2008): 501–8. http://dx.doi.org/10.1139/o08-139.

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The oxygen required to meet metabolic needs of all tissues is delivered by the erythrocyte, a small, flexible cell, which, in mammals, is devoid of a nucleus and mitochondria. Despite its simple appearance, this cell has an important role in its own distribution, enabling the delivery of oxygen to precisely meet localized metabolic need. When an erythrocyte enters in a hypoxic area, a signalling pathway is activated within the cell resulting in the release of ATP in amounts adequate to activate purinergic receptors on vascular endothelium, which trigger secretion of nitric oxide and other factors resulting in vasodilatation. Among other mechanisms, binding of deoxyhemoglobin to the cytoplasmic domain of the anion-exchange protein band 3 is probably involved in this pathway. The present study investigates the effect of amyloid β peptide exposure on this molecular mechanism. We report that deoxygenated human erythrocytes fail to release ATP following 24 h exposure to amyloid β peptide. Concurrently, amyloid β peptide induces caspase 3 activation. Preincubation of amyloid β peptide treated erythrocytes with a specific inhibitor of caspase 3 prevents amyloid-induced caspase 3 activation and restores the erythrocyte’s ability to release ATP under deoxygenated conditions. Since the activity of red cell phosphofructokinase, a key step in glycolytic flux, is not modified within the red cell following amyloid peptide exposure, it is likely that ATP release reduction is not dependent on glycolytic flux alterations. It has also been suggested that the heterotrimeric G protein, Gi, and adenylyl cyclase are downstream critical components of the pathway responsible for ATP release. We show that cAMP synthesis and ATP release are not failed in amyloid-peptide-treated erythrocytes in response to incubation with mastoparan 7 or forskolin plus 3-isobutyl-1-methyl xanthine, agents that stimulate cAMP synthesis. In conclusion, these results indicate that amyloid β peptide inhibits ATP release from deoxygenated erythrocytes by activating red cell caspase 3, suggesting a pathophysiologic role for vascular amyloid peptide in Alzheimer’s disease.

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Richmond, Terri D., Natasha Matthew, and Dwayne L. Barber. "Mutagenesis of Erythropoietin Receptor Cytoplasmic Lysines Uncouples Erythropoietin-Dependent Growth From Downstream Signal Transduction Cascades." Blood 114, no. 22 (November 20, 2009): 3611. http://dx.doi.org/10.1182/blood.v114.22.3611.3611.

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Abstract Abstract 3611 Poster Board III-547 Erythropoietin (EPO) is the primary cytokine regulator of erythropoiesis, stimulating growth, preventing apoptosis, and promoting differentiation of red blood cell progenitors. Fundamental to this action is the ability of EPO to bind to its cognate receptor on the cell surface, the EPO receptor (EPO-R), and activate the primary associated tyrosine kinase, JAK2. The critical importance of EPO, EPO-R and JAK2 to erythropoiesis is demonstrated by the fatal embryonic anemia that develops upon EPO, EPO-R or JAK2 deletion. Erythrocyte production relies heavily on precise control of EPO-mediated cell signaling cascades. The availability of EPO-R to EPO and the activation of subsequent signaling cascades are tightly regulated by the transit of mature EPO-R to the cell surface from the Golgi, and the mechanisms by which the receptor is internalized and down-regulated. Thus, small changes in plasma membrane EPO-R levels may be reflected in the intensity and duration of downstream signaling protein activation. EPO-R is rapidly ubiquitinated and down-regulated from the cell surface upon EPO stimulation. EPO-R ubiquitination relies on more than one E3 ubiquitin ligase, the U-box containing E3 ubiquitin ligase, p33Rul, and the RING finger E3 ubiquitin ligase SCFβTrcp. The discovery that EPO-R is ubiquitinated and degraded by the proteasome and lysosome lead us to examine the role of EPO-R ubiquitination on cell signaling and proliferation. We hypothesized that EPO-R ubiquitination would result in down-regulation of EPO-mediated signaling cascades. This study characterized which lysines determined EPO-R ubiquitination, and assessed the ability of EPO-R lysine mutants to support EPO-mediated signaling and proliferation. Surprisingly, substitution of all EPO-R cytoplasmic lysines (EPO-R K5R) abolished the ability of EPO-R to support EPO-mediated proliferation in BaF3 cells. However, EPO-dependent phosphorylation of EPO-R, AKT, PKB and STAT5 was detected, albeit at reduced levels. While ubiquitination defects commonly prolong receptor signaling, the kinetics of EPO-R K5R-mediated signaling pathways paralleled Ba/F3-EPO-R cells. Mutation of membrane proximal lysines, K256 and K276, resulted in decreased JAK2 phosphorylation and EPO-R phosphorylation and inhibited proliferation at 0.5U/ml EPO. However, the interaction between JAK2 and EPO-R was not affected. In addition, any EPO-R cytoplasmic lysine could support signaling and proliferation at physiologically elevated EPO concentrations demonstrating significant redundancy. The requirement of EPO-R cytoplasmic lysines to promote CFU-E formation is currently being evaluated by introduction of EPO-R mutants into fetal liver cells derived from EPO-R null mice. These results suggest that the EPO-R cytoplasmic lysines play a critical role in transmitting EPO-dependent growth signals within the cell. Disclosures: No relevant conflicts of interest to declare.

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Wang, Gang, Yan Huang, Ningning Zhang, Wenhu Liu, Changnan Wang, Xiaoyan Zhu, and Xin Ni. "Hydrogen Sulfide Is a Regulator of Hemoglobin Oxygen-Carrying Capacity via Controlling 2,3-BPG Production in Erythrocytes." Oxidative Medicine and Cellular Longevity 2021 (February 13, 2021): 1–16. http://dx.doi.org/10.1155/2021/8877691.

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Hydrogen sulfide (H2S) is naturally synthesized in a wide range of mammalian tissues. Whether H2S is involved in the regulation of erythrocyte functions remains unknown. Using mice with a genetic deficiency in a H2S natural synthesis enzyme cystathionine-γ-lyase (CSE) and high-throughput metabolomic profiling, we found that levels of erythrocyte 2,3-bisphosphoglycerate (2,3-BPG), an erythroid-specific metabolite negatively regulating hemoglobin- (Hb-) oxygen (O2) binding affinity, were increased in CSE knockout (Cse-/-) mice under normoxia. Consistently, the 50% oxygen saturation (P50) value was increased in erythrocytes of Cse-/- mice. These effects were reversed by treatment with H2S donor GYY4137. In the models of cultured mouse and human erythrocytes, we found that H2S directly acts on erythrocytes to decrease 2,3-BPG production, thereby enhancing Hb-O2 binding affinity. Mouse genetic studies showed that H2S produced by peripheral tissues has a tonic inhibitory effect on 2,3-BPG production and consequently maintains Hb-O2 binding affinity in erythrocytes. We further revealed that H2S promotes Hb release from the membrane to the cytosol and consequently enhances bisphosphoglycerate mutase (BPGM) anchoring to the membrane. These processes might be associated with S-sulfhydration of Hb. Moreover, hypoxia decreased the circulatory H2S level and increased the erythrocyte 2,3-BPG content in mice, which could be reversed by GYY4137 treatment. Altogether, our study revealed a novel signaling pathway that regulates oxygen-carrying capacity in erythrocytes and highlights a previously unrecognized role of H2S in erythrocyte 2,3-BPG production.

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Zhang, Yujin, Yingbo Dai, Jiaming Wen, Rodney E. Kellems, Michael R. Blackburn, Harinder S. Juneja, and Yang Xia. "Detrimental Role of Excess Adenosine-Mediated 2,3-Diphosphoglycerate Induction in Erythrocyte Sickling and Novel Mechanism-Based Therapies." Blood 114, no. 22 (November 20, 2009): 906. http://dx.doi.org/10.1182/blood.v114.22.906.906.

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Abstract Abstract 906 Introduction: Sickle cell disease (SCD) is a devastating inherited hemolytic disorder due to a single missense mutation in the β-globin gene resulting in the production of hemoglobin S (HbS). Despite our knowledge of the molecular defect in HbS, we remain unable to control HbS polymerization and erythrocyte sickling under hypoxic conditions, which are central to the pathophysiology of the disease. Methods and Results: Here we report that adenosine, a cellular metabolite that is well known to be induced under hypoxic/ischemic conditions, is increased in the blood circulation of patients with SCD and in SCD transgenic (Tg) mice. To our surprise, we found that adenosine contributes to hypoxia-induced sickling of cultured erythrocytes from humans with SCD. Next, to evaluate the pathogenic role of increased adenosine in erythrocyte signaling in vivo, we took advantage of SCD transgenic (Tg) mice, a well accepted animal model of SCD. Intriguingly, treatment of SCD Tg mice with polyethylene glycol-modified adenosine deaminase (PEG-ADA) enzyme therapy to lower circulating adenosine levels reduced sickling and attenuated multiple tissue damage (including lung, kidney, liver and spleen) seen in SCD Tg mice. These findings revealed a previously unrecognized role for excess adenosine in sickling and the progression to multiple life-threatening complications. Next, by screening erythrocytes for small metabolites that are induced by adenosine and may contribute to sickling, we determined that 2,3-diphosphoglycerate (2,3-DPG), an erythroid specific metabolite known to promote O2 release from Hb, is elevated in erythrocytes of both SCD Tg mice and humans. Using both pharmacological and genetic approaches, we demonstrated that adenosine-mediated 2,3-DPG induction is through A2B receptor signaling and that this signaling pathway is a major contributor to hypoxia-induced erythrocyte sickling. Conclusion and Significance: Overall, we have identified for the first time that elevated adenosine induces 2,3-DPG via A2BR signaling and contributes to sickling in both human and mouse with SCD. Thus, our studies provide strong support for the new concept that adenosine-mediated 2,3-DPG induction is beneficial for normal erythrocytes by promoting O2 release from Hb to hypoxic tissues. However, for SCD patients, the beneficial effect of adenosine-mediated 2,3-DPG induction becomes detrimental by inducing erythrocyte sickling due to 2,3-DPG mediated increased O2 release and increased deoxyHbS polymerization. Our findings reveal a novel therapeutic possibility to treat and prevent sickling and progression to multiple life-threatening complications by targeting on the adenosine signaling pathway. Disclosures: No relevant conflicts of interest to declare.

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Jones, Barry, Sharlene Adams, Glenn T. Miller, Michael I. Jesson, Takeshi Watanabe, and Barbara P. Wallner. "Hematopoietic stimulation by a dipeptidyl peptidase inhibitor reveals a novel regulatory mechanism and therapeutic treatment for blood cell deficiencies." Blood 102, no. 5 (September 1, 2003): 1641–48. http://dx.doi.org/10.1182/blood-2003-01-0208.

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Abstract In hematopoiesis, cytokine levels modulate blood cell replacement, self-renewal of stem cells, and responses to disease. Feedback pathways regulating cytokine levels and targets for therapeutic intervention remain to be determined. Amino boronic dipeptides are orally bioavailable inhibitors of dipeptidyl peptidases. Here we show that the high-affinity inhibitor Val-boro-Pro (PT-100) can stimulate the growth of hematopoietic progenitor cells in vivo and can accelerate neutrophil and erythrocyte regeneration in mouse models of neutropenia and acute anemia. Hematopoietic stimulation by PT-100 correlated with increased cytokine levels in vivo. In vitro, PT-100 promoted the growth of primitive hematopoietic progenitor cells by increasing granulocyte–colony-stimulating factor (G-CSF), interleukin-6 (IL-6), and IL-11 production by bone marrow stromal cells. Two molecular targets of PT-100 are expressed by stromal cells— CD26/DPP-IV and the closely related fibroblast activation protein (FAP). Because PT-100 was active in the absence of CD26, FAP appears to be the hematopoietic target for PT-100. Interaction of PT-100 with the catalytic site seems to be required because amino-terminal acetylation of PT-100 abrogated enzyme inhibition and hematopoietic stimulation. PT-100 is a therapeutic candidate for the treatment of neutropenia and anemia. The data support increasing evidence that dipeptidyl peptidases can regulate complex biologic systems by the proteolysis of signaling peptides.

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Journal articles on the topic 'Erythrocyte signaling pathways'

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