IL-3 and Retinoic Acid-Induced Myeloid Differentiation of Hematopoietic Progenitors Is Negatively Regulated by E3 Ubiquitin Ligase Rnf41 (FLRF).

Abstract

The proliferation and differentiation of hematopoietic cells is regulated by interaction of diverse group of cytokines with corresponding receptors. Regulation of cytokine signaling occurs at multiple levels, including the regulation of the amount of receptors present before and after ligand binding. However, the mechanisms that govern pre-determined steady-state amount of cytokine receptors are poorly understood. Previously we have reported that new E3 ubiquitin ligase Rnf41 (FLRF/Nrdp1) regulates cytokine-induced differentiation of hematopoietic progenitors through negative regulation of steady-state receptor levels. Increased levels of Rnf41 protein significantly attenuate differentiation of multipotent progenitors in response to IL-3, Epo and GM-CSF, due to a significant and constitutive decrease in the steady-state amount of IL-3, Epo and GM-CSF receptors. Immunoprecipitation and Western analysis of proteins from several types of hematopoietic progenitors has confirmed that Rnf41 protein associates with IL-3, Epo and GM-CSF receptors, and has shown that Rnf41-mediated down-regulation of receptors is independent of the ligand binding. Thus, by regulating steady-state amounts of IL-3, Epo and GM-CSF receptors Rnf41 could be maintaining optimal levels of signaling necessary for proper lineage commitment and differentiation of HSC and progenitors. Retinoic acid (RA) is known to augment IL-3 and GM-CSF induced differentiation of progenitors into myeloid lineages. Surprisingly, instead of improving the RA treatment further suppressed IL-3 and GM-CSF-induced myeloid differentiation of hematopoietic progenitors over-expressing Rnf41. The RA functions through RARα and RXR receptors, whihc act as transcription factors and interact with specific DNA targets as hetero- (RAR-RXR) or homodimers (RXR-RXR). Interestingly, Western analysis has shown that hematopoietic progenitors over-expressing Rnf41 exhibit a reduction in the steady-state levels of RARα receptors, whereas levels of RXR receptors remain unchanged. Moreover, the treatment of hematopoietic progenitors over-expressing Rnf41 with RA leads to even further decrease of RARα receptor levels. The transient over-expression of Rnf41 in BaF3 cell line also decreases steady-state levels of RARα receptors, while RXR receptor levels remain unchanged. Protein IP with α-Rnf41, α-RARα or α-RXR antibodies has shown that endogenous Rnf41 protein associates with RARα receptors in hematopoietic progenitors. Taken together, these results suggest that Rnf41 influences RA-mediated myeloid differentiation of hematopoietic progenitors by negatively regulating the levels of RARα receptors. Several studies have reported that IL-3 and GM-CSF-induced myeloid differentiation of hematopoietic progenitors leads to activation of RARa through induction of the Jak2/Stat5 pathway. These studies have defined a previously unknown cytokine-RAR interaction during myelopoiesis and suggested that RAR activation might be a critical downstream event following IL-3 and GM-CSF signaling during myeloid differentiation. Combined together the existing data suggest a model in which Rnf41 negatively regulates myeloid differentiation of hematopoietic progenitors by independently regulating steady state levels of IL-3, GM-CSF and RAR receptors, and thus impacting cytokine and RA signaling. Ongoing studies with RA agonists and antagonists are aimed at unraveling further the role of Rnf41 in regulation of RA signaling during progenitor cell differentiation.