E3 Ubiquitin Ligase Rnf41 (FLRF/Nrdp1) Regulates Steady-State Cytokine Receptor Levels in Multipotent Hematopoietic Progenitors.

Abstract Various cytokines regulate the development, proliferation, and differentiation of hematopoietic cells through interaction with their receptors. The mechanisms that govern pre-determined steady-state amount of cytokine receptors on the cell surface are poorly understood. Through screening of subtracted cDNA libraries prepared from mouse hematopoietic stem cells (HSC) and progenitors we have identified new RING finger protein Rnf41 (FLRF/Nrdp1). Rnf41 gene encodes a 317 aa protein with N-terminal C3HC4 RING finger domain, and TRAF-type zinc finger and coiled-coil domains. To elucidate its role in hematopoiesis we have over-expressed Rnf41 in a multipotent hematopoietic progenitor cell line EML. When cultured in the presence of SCF, Epo, Tpo, or IL-3 and GM-CSF, EML cells undergo multilineage differentiation, and give rise to erythroid, megakaryocytic, and granulocyte/macrophage progenitors. Over-expression of Rnf41 leads to (a) 3 to 5-fold decreased number of blast, erythroid, granulocyte/macrophage, and megakaryocytic progenitors being generated by EML cells, (b) significant and constitutive decrease in the amount of receptors for SCF, Epo, IL-3, GM-CSF and TPO being expressed by EML cells, and (c) significant increase in the amount of polyubiquitin chains associated with receptors. This down-regulation of receptors is independent of the ligand binding and receptor activation, and is also accompanied by delayed activation of MAPK and PI-3K signal transduction pathways and decreased phosphorylation levels of Erk and Akt. Furthermore, immunoprecipitation of proteins from EML cells and other hematopoietic progenitor cell lines has confirmed that Rnf41 protein associates with various cytokine receptors. The IL-3 and GM-CSF induced differentiation of EML cells is accompanied by enhanced transcriptional activation of retinoic acid receptor alpha (RARa), suggesting that RAR activation might be a critical downstream event following IL-3 and GM-CSF signaling during myeloid differentiation. Interestingly, although treatment with RA increases the number of progenitors being generated by wt EML cells, the same results in even further 2-fold decrease in the number of progenitors generated by EML cells over-expressing Rnf41. These data suggest that Rnf41 could be affecting the RAR pathway in EML cells either through down-regulation of IL-3 signaling pathway, thus decreasing activation of RAR, or by directly ubiquitinating endogenous RAR. In summary, Rnf41 binds to and marks for degradation a wide variety of hematopoietic cytokine receptors prior to ligand binding and receptor activation. Thus, Rnf41 is the first example of an E3 ubiquitin ligase whose function is to maintain predetermined amount of cell surface receptors. Through this Rnf41 could be maintaining optimal levels of signaling necessary for proper lineage commitment and differentiation of HSC and progenitors, while preventing oversignaling that could lead to leukemogenesis. Thus, over-expression of dominant negative forms of Rnf41 protein (lacking ring finger domain) could lead to receptor accumulation and cellular hyper-proliferation. In fact, the pilot experiments have shown that EML cells over-expressing truncated Rnf41 protein proliferate 3-fold faster than control EML cells. Ongoing experiments with over-expression of dominant negative forms of Rnf41 protein and RNAi inhibition of endogenous Rnf41 will reveal whether loss of or attenuated function of Rnf41 leads to receptor accumulation and cellular hyper-proliferation and differentiation.