Abstract
Elevated levels of the pro-inflammatory cytokine tumor necrosis factor-α (TNFα) inhibit erythropoiesis and cause anemia in patients with cancer and chronic inflammatory diseases. TNFα is also a potent activator of the sphingomyelinase (SMase)/ceramide pathway leading to ceramide synthesis and regulating cell differentiation, proliferation, apoptosis, senescence, and autophagy. Here we evaluated the implication of the TNFα/SMase/ceramide pathway on inhibition of erythropoiesis in human CD34+ hematopoietic stem/progenitor cells (CD34/HSPCs) from healthy donors. Exogenous synthetic C2- and C6-ceramide as well as bacterial SMase inhibited erythroid differentiation in erythropoietin-induced (Epo)CD34/HSPCs shown by the analysis of various erythroid markers. The neutral SMase inhibitor GW4869 as well as the genetic inhibition of nSMase with small interfering RNA (siRNA) against sphingomyelin phosphodiesterase 3 (SMPD3) prevented the inhibition by TNFα, but not the acid SMase inhibitor desipramine. Moreover, sphingosine-1-phosphate (S1P), a ceramide metabolite, restored erythroid differentiation, whereas TNFα inhibited sphingosine kinase-1, required for S1P synthesis. Analysis of cell morphology and colony formation demonstrated that erythropoiesis impairment was concomitant with a granulomonocytic differentiation in TNFα- and ceramide-treated EpoCD34/HSPCs. Inhibition of erythropoiesis and induction of granulomonocytic differentiation were correlated to modulation of hematopoietic transcription factors (TFs) GATA-1, GATA-2, and PU.1. Moreover, the expression of microRNAs (miR)-144/451, miR-146a, miR-155, and miR-223 was also modulated by TNFα and ceramide treatments, in line with cellular observations. Autophagy plays an essential role during erythropoiesis and our results demonstrate that the TNFα/neutral SMase/ceramide pathway inhibits autophagy in EpoCD34/HSPCs. TNFα- and ceramide-induced phosphorylation of mTORS2448 and ULK1S758, inhibited Atg13S355 phosphorylation, and blocked autophagosome formation as shown by transmission electron microscopy and GFP-LC3 punctae formation. Moreover, rapamycin prevented the inhibitory effect of TNFα and ceramides on erythropoiesis while inhibiting induction of myelopoiesis. In contrast, bafilomycin A1, but not siRNA against Atg5, induced myeloid differentiation, while both impaired erythropoiesis. We demonstrate here that the TNFα/neutral SMase/ceramide pathway inhibits erythropoiesis to induce myelopoiesis via modulation of a hematopoietic TF/miR network and inhibition of late steps of autophagy. Altogether, our results reveal an essential role of autophagy in erythroid vs. myeloid differentiation.
Highlights
Inflammatory cytokines cause anemia of cancer and chronic inflammatory diseases with consequences for the quality of life and lifespan
The effect of ceramide production was mimicked by treating EpoCD34/HSPCs with synthetic C2and C6-ceramide, bacterial SMase, and the bioactive sphingolipids derived from ceramide C2dihydroceramide and S1P
We show that exogenous C2- and C6-ceramide and bacterial SMase (bSMase)-induced endogenous ceramides prevented the expression of erythroid markers, erythropoiesis features, and miR-144/451 expression in EpoCD34/HSPCs to tumor necrosis factor-α (TNFα)
Summary
Inflammatory cytokines cause anemia of cancer and chronic inflammatory diseases with consequences for the quality of life and lifespan. Cytokines like tumor necrosis factor-α (TNFα) impair erythroid differentiation and perturbate hematopoietic homeostasis [1,2,3,4,5]. A network of receptors, cell signaling cascades, transcription factors (TFs), and microRNAs (miRs) time-dependently regulate hematopoiesis [6, 7]. TFs and miRs modulate genes involved in myeloid commitment. Major TF GATA-1 regulates erythropoiesis by modulating the negative regulator GATA-2 [8] and the erythroid miR-144/451 gene cluster [9]. Myeloid regulator PU. prevents erythropoiesis by binding to GATA-1. The GATA-1/PU. antagonism determines erythroid vs myeloid cell fate [10]. PU. regulates miR-146a, miR-155, and miR-223 genes associated with inhibition of erythropoiesis and granulomonocytic development [11,12,13]
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