Involving of the cytoplasmic region of leukemia inhibitory factor receptor α subunit, IL-6 related signal transducer-gp130 or Fas death domain for MAPK p42/44 activation in HL-60 cell with LIF or anti-Fas IgG
The chimeric receptors were prepared by exchanging the cytoplasmic region between leukemia inhibitory factor (LIF) receptor alpha subunit (gp190) and the other subunit-gp130 (190/130,130/190) and separately transduced into leukemia line HL-60 (to have the wild type subunit). The purpose is to investigate which subunit for activating MAPK p42/44 in leukemia cell while the cytoplasmic region homodimerization (190cyt-190cyt, 130cyt-130cyt) was induced by LIF. The results showed that MAPK p42/44 expression level after LIF stimulation 5 h was lower in the transformants with pED 130/190 (190cyt- 190cyt) (p < 0.01) and higher in the transformants with pED 190/130 (130cyt- 130cyt) (p < 0.05) than those in the parent cells. Meanwhile, MAPK p42/44 phosphorylation (Thr202/Tyr204) was ascended and the highest at 10 min in the 190/130 and descended in the 130/190. It suggests that gp130 activate MAPK p42/44 and gp190 indirectly regulate its expression and function. In order to analyses the relation of the subunit oligomerization and MAPK p42/44 we also prepared the recombination of the extracellular and transmembrane region of Fas and the cytoplasmic region of each LIFR subunit (Fas/190, Fas/130). After transduction into HL-60 with lipofection and induction by anti-Fas IgG, we found that MAPK p42/44 expression levels were lower in the Fas/190 than in the Fas/130 and parent cells (p < 0.01) and no difference between the Fas/130 and the wild type receptor. However, phospho-MAPK p42/44 were increased in the Fas/130 than the parent cells. It suggests that the oligomerization of the cytoplasmic regions of gp130 be potential to normally initiate MAPK p42/44 for the signal of HL-60 proliferation. We also determine that the separated oligomerization FasDD (no dimerization) can initiate the corresponding signal molecules, then regulate MAPK p42/44 expression and phosphorylation in leukemia cells.
"Leukemia inhibitory factor (LIF), as a member of the IL-6 family of cytokines, produces biological effects via binding to its receptor, which consists of a low-affinity LIF receptor α-chain (referred to as LIFRα) and a high-affinity subunit (referred to as gp130), both of which are shared with other IL-6 family cytokines (1). It has been reported that the 136-145-amino acid (aa) region of the distal C-terminal in cytoplasm, which contains 5-tyrosine residues (Y5) and several YXXQ motifs, is genetically conserved among gp130, LIFRα, and G-CSFR in numerous known cell lines (2,3). Thus, these motifs could initiate intracellular signaling by triggering LIFRα-gp130 heterodimerization or gp130-gp130/LIFRα-LIFRα homodimerization via Janus kinase-signal transducer and activator of transcription (Jak/STAT) and subsequently influence the growth or differentiation of leukemia cells (4). "
[Show abstract][Hide abstract] ABSTRACT: The distal cytoplasmic motifs of leukemia inhibitory factor receptor α-chain (LIFRα-CT3) can independently induce intracellular myeloid differentiation in acute myeloid leukemia (AML) cells by gene transfection; however, there are significant limitations in the potential clinical use of these motifs due to liposome-derived genetic modifications. To produce a potentially therapeutic LIFRα-CT3 with cell-permeable activity, we constructed a eukaryotic expression pcDNA3.0-TAT-CT3-cMyc plasmid with a signal peptide (ss) inserted into the N-terminal that codes for an ss-TAT-CT3-cMyc fusion protein. The stable transfection of Chinese hamster ovary (CHO) cells via this vector and subsequent selection by Geneticin resulted in cell lines that express and secrete TAT-CT3-cMyc. The spent medium of pcDNA3.0-TAT-CT3-cMyc-transfected CHO cells could be purified using a cMyc-epitope-tag agarose affinity chromatography column and could be detected via SDS-PAGE, with antibodies against cMyc-tag. The direct administration of TAT-CT3-cMyc to HL-60 cell culture media caused the enrichment of CT3-cMyc in the cytoplasm and nucleus within 30 min and led to a significant reduction of viable cells (P < 0.05) 8 h after exposure. The advantages of using this mammalian expression system include the ease of generating TAT fusion proteins that are adequately transcripted and the potential for a sustained production of such proteins in vitro for future AML therapy.
Brazilian journal of medical and biological research = Revista brasileira de pesquisas medicas e biologicas / Sociedade Brasileira de Biofisica ... [et al.] 06/2012; 45(10):913-20. DOI:10.1590/S0100-879X2012007500101 · 1.01 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A chimeric receptor (130/190) containing the cytoplasmic region of leukemia inhibitory factor receptor alpha subunit (LIFRalpha, or gp190) and the extracellular transmembrane region of gp130 was generated. Expressed of the 130/190 chimera in HL-60 cells to induced the homodimerization of the cytoplasmic domains (190cyt-190cyt) with whole LIFRalpha subunit on HL-60 cells in response to LIF. Expression and activation of the signal transducer and activator of transcription factor-3 (Stat3) and inhibition of leukemia cell proliferation were evaluated in cells transfected with this chimeric molecule. Increased tyrosyl phosphorylation of Stat3 at Tyr705 was detected after 10 min LIF treatment in cells transfected with either the 130/190 or the wild type receptor. Cell proliferation was decreased upon LIF treatment in both cell types. However, expression of the C-terminal region of the cytoplasmic region of LIFRalpha subunit (190CT) in HL-60 cells resulted in lower levels of Stat3 phosphorylation induction by LIF and cell proliferation was unaffected. Immunohistochemical staining indicated an inverse correlation between Cdc25B expression and the levels of phospho-Stat3 in 190CT and 130/190 cells. Expression of CD15, a cell differentiation marker, was lower in 190CT than in 130/190 cells. Together, these results suggest that homodimerization of the 190 cytoplasmic region promotes the Tyr 705 phosphorylation, which correlates with the inhibition of proliferation and stimulation of differentiation in HL-60 cells. Our results also suggest a signal link between Stat3 and Cdc25B.
[Show abstract][Hide abstract] ABSTRACT: The leukemia inhibitory factor (LIF) affects multiple types of leukemia cells in vitro through the functional LIF receptor (LIFR), which comprises a complex of the LIFR α-chain (LIFR α) and gp130. As Jak2/STAT3 has been proven to be a significant mediator in the LIF-induced differentiation of promyeloid leukemia cells, we constructed a recombinant vector, pcDNA3.0-CT3 (containing the structurally conserved triple YXXQ motifs of LIFR α, termed LIFR α-CT3), and its specific tyrosine-mutated counterpart, pcDNA3.0-MUT, to determine the sites and examine the corresponding mechanisms involved in STAT3 phosphorylation. We found that the triple YXXQ motifs of LIFR α-CT3 are capable of up-regulating phosphorylated levels of STAT3 in a Jak2-independent manner prior to the induction of myeloid differentiation by LIFR α-CT3 in the human promyeloid cell line HL-60. By specifically blocking Jak2 using the AG-490 inhibitor, we observed that the LIFR α-CT3 group of HL-60 cells still demonstrated up-regulation of phosphorylated STAT3 and this up-regulation could result in the myeloid differentiation of HL-60 cells. These results may shed light on acute promyeloid leukemia therapy in future clinical practice.
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