P2 Receptor Antagonists Rescue Defective Heme Content in an In Vitro SLC25A38-Associated Congenital Sideroblastic Anemia Cell Model

Abstract

Mutations in the SLC25A38 gene are responsible for the second most common form of congenital sideroblastic anemia (CSA), a severe condition for which no effective treatment exists. We developed and characterized a K562 erythroleukemia cell line with markedly reduced expression of the SLC25A38 protein (A38-low cells). This model successfully recapitulated the main features of CSA, including reduced heme content and mitochondrial respiration, increase in mitochondrial iron, ROS levels and sensitivity to oxidative stress. Notably, our study uncovered a new role for extracellular pyridoxal 5'-phosphate (PLP) and other P2 receptor antagonists in rescuing the altered parameters of A38-low cells (for example, the heme content of the A38-low cells was increased from about 50% to about 80% by the P2 receptor antagonists treatment compared with the value of the controls). These findings suggest that targeting P2 receptors could represent a promising therapeutic approach for SLC25A38-associated CSA.

Keywords: P2 receptors; SLC25A38; congenital sideroblastic anemia (CSA); heme biosynthesis; iron dyshomeostasis; mitochondrial carriers; oxidative stress; pyridoxal 5′-phosphate.

Figure 1

(A) Expression level of SLC25A38 in A38-low and K562 WT cell lines. Total cellular proteins (50 µg) from the indicated cells were probed by a Western blot analysis for SLC25A38 expression. β-ATP synthase and free staining gel served as loading control. (B) Heme content in K562 cell lines. Heme content was determined in K562 cell lines from wild type (WT) (white), A38-low (blue) and A38-low cells transfected with pcDNA3.1-SLC25A38 plasmid (pA38-low) (grey) (** p < 0.01, *** p < 0.001, Student’s t test; n = 5). (C) Mitochondrial LIP in K562 cell lines. A38-low and WT cells were stained with 5 µM Mito-FerroGreen and analyzed using an Attune Acoustic Focusing Cytometer. (D) ROS levels in K562 WT and A38-low cells. Representative histograms illustrating the cellular and mitochondrial ROS levels were determined by cytofluorimetric analysis using 2′,7′-dichlorodihydrofluorescein diacetate (DCF-DA) and dihydrorhodamine 123 (DHR), respectively. The short black line in the figure highlights the populations with elevated oxidative stress. (E) PPIX contents in K562 WT (white) and A38-low cells (blue). PPIX was determined in total cellular extracts by a fluorescence method [21]. The fluorescence values were normalized to mg/mL of each sample. PPIX content is expressed as the control-related fold change (*** p < 0.001, Student’s t test; n = 4). (F) Oxidative stress sensitivity. Cell viability of indicated cells, assessed through the resazurin assay, was determined following a 48 h treatment with H₂O₂ at the indicated concentrations. The data are presented as a percentage of the control group (untreated), (** p < 0.01; *** p < 0.001, two-way ANOVA test; n = 3).

Figure 2

Effect of folic acid with glycine and B6 vitamers on A38-low cells. (A) Heme content, assessed in K562 WT, as well as in the A38-low cell line untreated (NT) or treated for 48 h with a mixture of 1 mM folic acid and 100 mM glycine (F/G), or with pyridoxal 5′-phosphate (PLP), pyridoxine (PN), pyridoxal (PL), and pyridoxamine (PM) at the concentrations of 0.1 mM. The values are expressed as the fold change related to the control cells. Number of replicates: 4 or 5. (B) Cellular and mitochondrial ROS levels in A38-low cells untreated (−) or treated with 0.1 mM PLP (+), expressed as the fold change in median fluorescence intensity as determined by cytofluorimetric analysis, using 2′,7′-dichlorodihydrofluorescein diacetate (DCF-DA) and dihydrorhodamine 123 (DHR), respectively. Number of replicates: 3. (C) State III respiration, in the presence of malate, pyruvate, glutamate, succinate and ADP, assessed in WT and in A38-low cell lines. Additionally, A38-low cells were treated with 0.1 mM PLP for 48 h. Comparative measurements were conducted in a pairwise manner against their respective control counterpart (* p < 0.05; ** p < 0.01; *** p < 0.001; Student’s t-test; n = 4 or 5).

Figure 3

Pyridoxal 5′-phosphate content in WT and A38-low cell lines. (A) Representative histograms illustrating the intracellular pyridoxal 5′-phosphate (PLP) content, determined by cytofluorimetric analysis using the AcRAB6 probe, were obtained for the K562 WT and A38-low cell lines. (B) Epifluorescence images showing untreated K562 WT and A38-low cells (NT), along with A38-low cells treated with 50 µM pyridoxine (PN), pyridoxamine (PM), and pyridoxal (PL), and stained with AcRAB6 for 12 h as reported [22].

Figure 4

Involvement of the P2 receptor in the PLP-mediated effect. Heme content was determined in total cell extracts from K562 WT (white circle) and A38-low cells (blue circle) after treatment with pyridoxal 5′-phosphate (PLP), pyridoxal (PL), pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid (PPADS), and suramin (SUR) at the indicated concentrations (µM) for 48 h. Data were indicated as the control-related ratio (* p < 0.05; ** p < 0.01; Student’s t-test untreated-related; n = 4 or 5).