Evidences of the PI5P Increasing the Expression of HBG and Gamma Globin Concentrations

Efforts have been made to identify modulators of increasing fetal hemoglobin (HbF - α2ɣ2) that may act as promising targets for hemoglobinopathies, such as in sickle cell disease and beta thalassemia. The inositide PI5P is a second messenger from the inositol pathway that comprehends in the main phosphorylation target of the enzyme phosphatidylinositol-4-phosphate-5-kinase-II-alpha (PIPKIIα), found in high concentrations in the erythrocyte. Some evidences have postulated a relation between this enzyme and the expression of the hemoglobin (Hb) genes (1,2). More recently, PI5P was found to stabilize UHRF1 protein (3), which has been reported as a possible repressor of HBG (from ɣ globin gene), in the switch from ɣ to β in the transition HbF- HbA (4). To evaluate the effects of the inositide PI5P (substrate of PIPKIIα) in globins, a preliminary time course experiment was performed. KU812 cells (ATCC CRL-2099), which express all three globin genes (HBA, HBB and HBG), were treated with 1μM of PI5P (Thermo Fischer Scientific, USA) in triplicate (4, 8 and 12 hours of treatment) - time 0h used as control. Gene expression and protein concentrations were determined by qPCR - SYBR Green Master Mix amplification and detection kit (Thermo Fischer Scientific, USA, in equipment StepOnePlus Real Time PCR System, Applied Biosystems - USA) using 2DDCT method with the ACTB and GAPDH genes as internal controls - and immunoblotting. Primers were design according to Zacariotto et al (2). No additional phosphate was imputed into the cell culture and the ATP consumption was monitored by ATP Determination Kit (Thermo Fisher Scientific, EUA). PI5P treatment resulted in significant increasement of HBG RNA transcripts (p= 0.001; 8 hours / treatment) and decrease of PIP4K2A (from PIPKIIα enzyme) (p=0.02; 12 hours / treatment), with no other significant changes into globin genes (HBA and HBB). At the protein level, there was a prominent increase in ɣ globin concentrations according to time course. No other consistent change in the concentrations of PIPKIIα or globin chains were observed. Due to protein similarities between the subfamilies of phosphatidylinositol-phosphate kinases (PIPKins) (5), the gene expression of these enzymes was also monitored by qPCR. There was a significant reduction of PIP5K1A (from PIPKIα enzyme) (p=0.01 - 4h; p=0.01 - 8h, and p=0.009 - 12h) as well as PIP4K2B (from PIPKIIβ enzyme) (p=0.03 - 4 hours/ treatment). No significant changes were observed in the other PIPKins (PIPKIβ, PIPKIɣ, PIPKIIɣ and PIPKIII). Considering that a significant consumption of intracellular ATP was observed in time course (p=0.006) it is possible to infer that the high concentrations of PI5P have shifted the inositol pathway resulting in the downregulation of its own PIPKin genes and culminating into the upregulation of HBG, reflected into the protein level. The mechanisms involving the inositide as second messenger, the role of PIPKins or other genes into the modulation of hemoglobin genes, particularly in HBG, should be further investigated. However, despite preliminary, these results reinforce the involvement of the PIPKins and its molecular targets (mainly PIPKIα and PI5P) in globin gene regulation and could represent a promising target for future therapeutic target for hemoglobinopathies. Financial Support: Fapesp, CNPq, CAPES, Faepex-Unicamp.

1.Wenning MR, Mello MP, Andrade TG, et al. PIP4KIIA and beta-globin: transcripts differentially expressed in reticulocytes and associated with high levels of Hb H in two siblings with Hb H disease. Eur J Haematol. 2009; 83: 490-3.

2.Zaccariotto TR, Lanaro C, Albuquerque DM, Santos MN, Bezerra MA, Cunha FG, et al. Expression profiles of phosphatidylinositol phosphate kinase genes during normal human in vitro erythropoiesis. Genet Mol Res. 2012; 11: 3861-8.

3.Gelato KA, Tauber M, Ong MS, Winter S, et al. Accessibility of different histone H3-binding domains of UHRF1 is allosterically regulated by phosphatidylinositol 5-phosphate. Mol Cell. 2014;54(6):905-19.

4.Ruopeng Feng, Phillip A Doerfler, Yu Yao XT, et al. The DNA Methylation Maintenance Protein UHRF1 Regulates Fetal Globin Expression Independent of H BG Promoter DNA Methylation. Blood. 2018;132:410.

5.Heck JN, Mellman DL, Ling K, et al. A conspicuous connection: structure defines function for the phosphatidylinositol-phosphate kinase family. Crit Rev Biochem Mol Biol. 2007; 42: 15-39.