Pivotal role for the NFIL3/E4BP4 transcription factor in interleukin 3-mediated survival of pro-B lymphocytes

Abstract

The E2A-HLF (hepatic leukemia factor) oncoprotein, generated in pro-B lymphocytes by fusion of the trans-activation domain of E2A to the basic region͞ leucine zipper (bZIP) domain of HLF, functions as an anti-apoptotic transcription factor in leukemic cell transformation. When introduced into interleukin 3 (IL-3)-dependent mouse pro-B lymphocytes, E2A-HLF prevents apoptosis induced by growth factor deprivation, suggesting that IL-3 mediates cell survival through activation of a transcription factor whose activity can be constitutively replaced by the chimeric oncoprotein. We considered four bZIP transcription factors as candidates for this putative IL-3-regulated factor, each of which binds avidly to the DNA consensus sequence recognized by E2A-HLF and is related to the Caenorhabditis elegans CES-2 (cell death specification protein) neuron-specific mediator of cell death. The expression and binding activity of the Nfil3 protein (also called E4bp4), but not of Hlf, Dbp, or Tef, was found to be regulated by IL-3 in mouse pro-B cell lines (Baf-3 and FL5.12). Northern blot analysis showed that Nfil3͞E4bp4 is regulated as a ''delayed-early'' IL-3-responsive gene, requiring de novo protein synthesis. In the absence of IL-3, enforced expression of the human NFIL3͞E4BP4 cDNA promoted the survival but not the growth of IL-3-dependent pro-B cells. Our results implicate NFIL3͞E4BP4 (nuclear factor regulated by IL-3͞adenovirus E4 promoter binding protein) in a distinct growth factor-regulated signaling pathway that is responsible for the survival of early B-cell progenitors, and whose alteration by E2A-HLF leads to childhood B lineage leukemia.

Calcium-dependent upregulation of E4BP4 expression correlates with glucocorticoid-evoked apoptosis of human leukemic CEM cells

“…Other homologous PAR domain lacking bZIP genes include the C. elegans ces-2 (cell death selector) gene, which promotes neuronal cell death, the Drosophila vrille gene, which induces apoptosis in embryonic dorsal epidermis, and genes 8 and 9 in X. laevis, which contribute to tail resorption [13,14]. Based on these homologous genes, one might expect E4BP4 to be pro-apoptotic; however, depending of the cell type and downstream effectors, E4BP4 has been shown to promote cell survival [15,16], induce apoptosis [17], or promote parathyroid hormone-mediated catabolism in osteoblasts [11]. In human Blymphocytes, E4BP4 is upregulated by IL-3 (hence the alternative name: Nuclear factor regulated by IL3; NFIL3) and promotes survival of IL-3-dependent cells [15].…”

“…Based on these homologous genes, one might expect E4BP4 to be pro-apoptotic; however, depending of the cell type and downstream effectors, E4BP4 has been shown to promote cell survival [15,16], induce apoptosis [17], or promote parathyroid hormone-mediated catabolism in osteoblasts [11]. In human Blymphocytes, E4BP4 is upregulated by IL-3 (hence the alternative name: Nuclear factor regulated by IL3; NFIL3) and promotes survival of IL-3-dependent cells [15]. Similarly, in rat motoneurons, E4BP4 serves as a survival factor, and is expressed at high levels in those neurons that escape natural apoptosis [16].…”

“…Reagents for RT-PCR, including M-MLV reverse transcriptase, oligo(dT) 15 primer, RNasin ® Ribonuclease inhibitor, dNTP mix, and Taq DNA polymerase were purchased from Promega Life Sciences (Madison, WI). Calcium Green™-1 AM, and propidium iodide were purchased from Molecular Probes™/Invitrogen (Carlsbad, CA).…”

“…Approximately 1 × 10 7 cells were harvested, washed once with chilled PBS, and RNA was extracted using the TRIzol reagent (Invitrogen Life Technologies, La Jolla, CA). Five micrograms of total RNA was reverse transcribed at 42 °C for 2 h in the presence of 0.5 μg oligo(dT) 15 , 1 μl (∼200 U) M-MLV reverse transcriptase, 0.5 mM dNTP mix, and 100 U RNase inhibitor. A 606 bp E4BP4-specific DNA fragment corresponding to nucleotides 709-1314 of the E4BP4 cDNA (GenBank Accession No.…”

“…Genes closely related to mammalian E4BP4 include genes 8 & 9 of Xaenopus laevis, ces-2 of Caenorhabditis elegans, and vrille of Drosophila melanogaster, all of which are involved in pro-apoptotic events such as tail resorption, neuronal cell death, and activation of pro-apoptotic signaling molecules [13,14]. In contrast to its evolutionarily conserved function as a promoter of cell death, E4BP4 has been suggested to protect growth factor-dependent B-lymphocytes from apoptosis caused by interleukin-3 deprivation [15]. E4BP4 has been shown to regulate motoneuron growth and survival during neuronal development [16].…”