TARGETING GAMMA GLOBIN SYNTHESIS: THE ACUTE EFFECTS OF PIPKIIA INHIBITION AND PTDIN5P STIMULATION IN MYELOID CELL LINES K562 AND KU812

Abstract

Introduction: Phosphoinositide (PtdIns)-mediated cellular signaling and their regulatory proteins (phosphatidylinositol phosphate kinases - PIPKins) are involved in many cell functions. The Hemoglobinopathies Laboratory hypothesizes a relationship between PIPKins, particularly PIPKIIa, and the regulation of globin gene expression in a panel of cells. Recent data suggest a possible mechanism for regulating the fetal hemoglobin (HbF-α2γ2) to adult hemoglobin (HbA-α2β2) transition mediated by these enzymes. Objectives: To investigate the production of Hb gamma (γ) chains after PIPKIIa inhibition and after stimulating its activity by adding its main substrate, PtdIns5P. Materials and methods: Immortalized myeloid cells K562 (ATCC CCL-243) and KU812 (ATCC CRL-2099) were treated with 1 μM of PIPKIIa inhibitory drug BAY-091 (MedChem Express, USA) or 1 μM of synthetic PtdIns5P (Thermo Fisher, USA) and BAY-091 + PtdIns5P for 24 hours following dose-response assays for IC-50 calculation. All experiments were performed in biological triplicate (n = 3). Cells were collected, fixed, permeabilized (Cell Fixation & Cell Permeabilization Kit - Thermo Fisher, USA), and incubated with anti-HbF antibody (Thermo Fisher, USA). HbF (γ chains) positive cells were analyzed by flow cytometry (BD FACSCalibur). Qualitative (% total labeled cells) and quantitative (geometric mean of individual fluorescence intensity) analyses were performed by FlowJo program (BD Biosciences, USA). Statistical analyses were conducted using SPSS (IBM, USA) with one-way ANOVA and multiple comparisons with Tukey's test (α < 0.05). Results and discussion: Inhibition of PIPKIIa resulted in a consistent qualitative reduction (% total positive cells) of γ chains (p = 0.01) in K562 cells, as well as with the inhibition + stimulation with PtdIns5P (p = 0.04). No significant differences were observed after treatment with PtdIns5P alone (p = 0.08). The same pattern was observed quantitatively: intracellular reduction of γ chains (p = 0.01) and inhibition + stimulation (p = 0.02). In KU812 cells, stimulation with PtdIns5P resulted in qualitative (p = 0.01) and quantitative (p = 0.03) reduction of γ chains and a quantitative reduction with inhibition + stimulation (p = 0.04). Although individual stimulation with PtdIns5P did not influence γ chain production in K562 cells, data suggest that PIPKIIa activity and PtdIns5P might modulate γ chain production of Hb in these cell lines, as observed in KU812 cells following individual stimulation. Both cell lines showed γ chain reduction after BAY-091 + PtdIns5P treatment, suggesting a synergistic effect where inhibition of PIPKIIa by BAY-091 potentiates PtdIns5P's action by reducing its conversion into other phosphoinositides, increasing its bioavailability, possibly affecting chromatin structure, as demonstrated in previous literature. Conclusion: Inhibition of PIPKIIa activity, as well as its combination with PtdIns5P stimulation, reduces the production of Hb γ chains in K562 and KU812 cells, suggesting an important role of this enzyme in globin gene regulation. Financial support: Fapesp, CAPES, CNPq, Funcamp, and Faepex.