casein-kinase-ii and Anemia--Sickle-Cell

Increased endothelin-1 (ET-1) levels, disordered thiol protein status, and erythrocyte hydration status play important roles in sickle cell disease (SCD) through unresolved mechanisms. Protein disulfide isomerase (PDI) is an oxidoreductase that mediates thiol/disulfide interchange reactions. We provide evidence that PDI is present in human and mouse erythrocyte membranes and that selective blockade with monoclonal antibodies against PDI leads to reduced Gardos channel activity (1.6±0.03 to 0.56±0.02 mmol·10(13) cell(-1)·min(-1), P<0.001) and density of sickle erythrocytes (D50: 1.115±0.001 to 1.104±0.001 g/ml, P=0.012) with an IC50 of 4 ng/ml. We observed that erythrocyte associated-PDI activity was increased in the presence of ET-1 (3.1±0.2 to 5.6±0.4%, P<0.0001) through a mechanism that includes casein kinase II. Consistent with these results, in vivo treatment of BERK sickle transgenic mice with ET-1 receptor antagonists lowered circulating and erythrocyte associated-PDI activity (7.1±0.3 to 5.2±0.2%, P<0.0001) while improving hematological parameters and Gardos channel activity. Thus, our results suggest that PDI is a novel target in SCD that regulates erythrocyte volume and oxidative stress and may contribute to cellular adhesion and endothelial activation leading to vasoocclusion as observed in SCD.

Topics: Anemia, Sickle Cell; Animals; Antibodies, Monoclonal; Calcimycin; Calcium Ionophores; Casein Kinase II; Cell Membrane; Endothelin A Receptor Antagonists; Enzyme Inhibitors; Erythrocyte Volume; Erythrocytes; Humans; Intermediate-Conductance Calcium-Activated Potassium Channels; Mice; Oxidative Stress; Protein Disulfide-Isomerases; Sulfhydryl Compounds

Lutheran (Lu) blood group and basal cell adhesion molecule (B-CAM) antigens reside on two glycoprotein (gp) isoforms Lu and Lu(v13) that belong to the Ig superfamily and differ only by the size of their cytoplasmic tail. Lu/B-CAM gps have been recognized as laminin alpha5 receptors on red blood cells and epithelial cells in multiple tissues. It has been shown that sickle red cells exhibit enhanced adhesion to laminin alpha5 when intracellular cAMP is up-regulated by physiological stimuli such as epinephrine and that this signaling pathway is protein kinase A- and Lu/B-CAM-dependent. In this study, we analyzed the relationship between the phosphorylation status of Lu/B-CAM gps and their adhesion function to laminin alpha5. We showed that Lu isoform was phosphorylated in sickle red cells as well as in erythroleukemic K562 and epithelial Madin-Darby canine kidney cells and that this phosphorylation is enhanced by different stimuli of the PKA pathway. Lu gp is phosphorylated by glycogen synthase kinase 3 beta, casein kinase II, and PKA at serines 596, 598, and 621, respectively. Alanine substitutions of serines 596 and 598 abolished phosphorylation by glycogen synthase kinase 3 beta and casein kinase II, respectively, but had no effect on adhesion of K562 cells to laminin under flow conditions. Conversely, mutation of serine 621 prevented phosphorylation by PKA and dramatically reduced cell adhesion. Furthermore, stimulation of K562 cells by epinephrine increased Lu gp phosphorylation by PKA and enhanced adhesion to laminin. It is postulated that modulation of the phosphorylation state of Lu gp might be a critical factor for the sickle red cells adhesiveness to laminin alpha5 in sickle cell disease.

Topics: Adrenergic beta-Antagonists; Alanine; Amino Acid Sequence; Anemia, Sickle Cell; Animals; Blotting, Western; Butoxamine; Casein Kinase II; Cell Adhesion; Cell Adhesion Molecules; Cell Line; Colforsin; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Cytoplasm; Dogs; Epinephrine; Epithelial Cells; Erythrocytes; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Glycoproteins; Humans; Immunoprecipitation; K562 Cells; Kidney; Laminin; Lutheran Blood-Group System; Molecular Sequence Data; Mutation; Neoplasm Proteins; Phosphorylation; Protein Structure, Tertiary; Recombinant Proteins; Serine; Signal Transduction; Up-Regulation