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.

To investigate the production of Hb gamma (γ) chains after PIPKIIa inhibition and after stimulating its activity by adding its main substrate, PtdIns5P.

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).

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.

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.

Fapesp, CAPES, CNPq, Funcamp, and Faepex.