Department of Pharmacy, Research Institute of Pharmaceutical S...

Elevated levels of the pro-inflammatory cytokine tumor necrosis factor- TNF inhibit erythropoiesis and cause anemia in patients with cancer and chronic inflammatory diseases TNF 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 TNF 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 TNF but not the acid SMase inhibitor desipramine Moreover sphingosine-1-phosphate S1P a ceramide metabolite restored erythroid differentiation whereas TNF 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 TNF -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 TNF and ceramide treatments in line with cellular observations Autophagy plays an essential role during erythropoiesis and our results demonstrate that the TNF neutral SMase ceramide pathway inhibits autophagy in EpoCD34 HSPCs TNF -and ceramide-induced phosphorylation of mTOR S2448 and ULK1 S758 inhibited Atg13 S355 phosphorylation and blocked autophagosome formation as shown by transmission electron microscopy and GFP-LC3 punctae formation Moreover rapamycin prevented the inhibitory effect of TNF 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 TNF 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

Department of Pharmacy, Research Institute of Pharmaceutical Sciences, College of Pharmacy, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul 08826, South Korea

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Sphingolipid-mediated inflammatory signaling leading to autophagy inhibition converts erythropoiesis to myelopoiesis in ...