GATA1-Dependent Ceramide Homeostasis Controls Cytokine Signaling and Erythroid Cell Function

Our multi-omic studies in cultured erythroblast cells (G1E-ER-GATA1) revealed that GATA1 regulates the synthesis and/or transport of small molecules, including heme, metal ions, and nucleosides (Liao et al. Cell Rep. 2020, Tanimura et al. Dev. Cell 2018, Zwifelhofer et al. PLoS Genet. 2020). These cells are GATA1-null proerythroblast-like cells stably expressing a β-estradiol-induced allele encoding GATA1 fused to the hormone binding domain of estrogen receptor. Analysis of the GATA1-regulated transcriptome and proteome in G1E-ER-GATA1 cells revealed multiple differentially-expressed components involved in sphingolipid biosynthesis. Sphingolipids are a group of signaling lipids, including ceramides, which regulate cell proliferation and apoptosis, and sphingosine-1-phosphate that controls vascular homeostasis. One of these genes, Degs1, encodes dihydroceramide desaturase (DES) that converts dihydroceramides to ceramides. GATA1 upregulates Degs1 mRNA expression by 12.2-fold and DEGS1 protein by 4.6-fold. GATA1 occupies the promoter and intronic sequences of murine Degs1 locus, suggesting that Degs1 is a GATA1 target gene. Quantitative lipidomics revealed that intracellular ceramide, together with sphingosine and sphingosine-1-phosphate, increased during erythroid maturation, whereas dihydroceramide did not change significantly.

To determine the functional significance of GATA1-induced Degs1 expression, we chemically inhibited DES activity with 4-hydroxyphenyl retinamide (4HPR) in β-estradiol-induced G1E-ER-GATA1 cells and quantified sphingolipids using quantitative lipidomics. 4HPR increased dihydroceramide 7.8-fold at 24 h and 14-fold at 48 h, while ceramide was reduced 1.7 and 1.9-fold, respectively, which was associated with reduced cell growth, hemoglobinization, and GATA1-dependent gene activation. Treating primary fetal liver HSPCs with 4HPR significantly reduced the number of CFU-E by 4.8-fold (p<0.0001) and BFU-E by 4.8-fold (p<0.0001), but not CFU-GM. Furthermore, treating the cells with C6-dihydroceramide (C6-dhCer) or C6-ceramide (C6-Cer), which elevates intracellular ceramides, strongly impaired erythroid, but not myeloid, colony formation. Thus, disrupting dihydroceramide or ceramide homeostasis is deleterious to mouse erythroid progenitor cells.

To elucidate the mechanistic underpinnings of the erythroid cell hypersensitivity to (dh)ceramides, we asked if disrupting ceramide homeostasis impacts stem cell factor (SCF) and erythropoietin (Epo) signaling, which are essential for erythroid cell proliferation, survival, and differentiation. Inhibiting DES with 4HPR or treating the cells with C6-dhCer or C6-Cer significantly reduced SCF-dependent induction of p-AKT and p-ERK and Epo-dependent induction of p-AKT, p-ERK, and p-STAT5, suggesting that both SCF and Epo signaling are inhibited by (dh)ceramides. Short-term C6-Cer treatment impaired Epo-dependent activation of p-AKT and p-ERK, but not p-STAT5, suggesting a differential sensitivity of downstream signaling pathways. In addition, C6-Cer reduced SCF-induced p-AKT without affecting activation of its receptor, c-Kit, indicating that ceramides inhibit cytokine signaling post-receptor activation. Since ceramide activates protein phosphatase PP2A in vitro, we asked if PP2A mediates ceramide-dependent inhibition of cytokine signaling. PP2A inhibition with okadaic acid rescued ceramide-dependent reduction of p-AKT and p-ERK. Furthermore, two distinct PP2A activators, FTY-720 and DT-061, impacted SCF and Epo-dependent signaling similar to C6-Cer, suggesting that ceramide inhibits cytokine signaling via activation of PP2A. Finally, we demonstrated that Epo-dependent GATA1 phosphorylation is impaired by 4HPR or C6-Cer. These findings establish a paradigm in which GATA1 controls sphingolipid biosynthetic enzymes and the generation of regulatory lipids that restrict cytokine signaling, thereby impacting erythrocyte development and function. As inflammatory mediators implicated in anemia of inflammation elevate dihydroceramides and ceramides, we propose that the integrated transcriptional, metabolic and signaling mechanism has broad importance in erythroid biology and pathology.

No relevant conflicts of interest to declare.