γ-secretase Can Cleave Gp190 To Inhibit Human Leukemia Through Activating STAT3 Signaling Pathway

Leukemia is a fatal disease that threats human health, many researches focus on the pathopoiesis of Leukemia with the hope of finding an effective treatment for Leukemia. Recent study showed thatγ-secretase inhibitor can suppress cell proliferation by inhibiting Notch1 signaling, one of the substrates ofγ-secretase.γ-secretase is a multiprotein complex responsible for the intramembrane cleavage of Notch, amyloid precursor protein(APP),CD44 and numerous other type-I transmembrane receptors,indicating that it normally serves as a mediator of diverse signaling pathways.γ-secretase consists of 4 subunits , Presenilin(PS), Nicastrin(NCT), Anterior pharynxdefective-1 (Aph-1) and Presenilin enhancer 2(Pen-2), PS is the active site of the protease,which is cleaved into N-terminal and C-terminal fragments(NTF and CTF) that remain associated,and these heterodimers appear to be the biologically active form of the protein. Aph-1 and pen-2 are essential for PS endoproteolysis andγ-secretase function.NCT, discovered via its tight association with PS, is―substrate receptor‖ofγ-secretase,and its overexpression can enhanceγ-secretase activity leading to more Aγproduction,which is the pathogenic material of Alzheimer disease.Notch gene exists in a variety of species and their family members are highly conserved in evolution, Notch-mediated signaling pathway is involved in embryonic development, blood cell development, tumor formation, differentiation and proliferation of leukemia cell and many other pathophysiological processes. Notch is a substrate ofγ-secretase enzyme and 4 genes respectively encoding Notch1-4 was found in mammals. Notch1 was cleavaged by ADAM metalloproteinases andγ-secretase into intracellular membrane fragments ICN1 (intracellular domain Notch1), which was released and transfered into the nucleus.With the recruitment of MAML, CSL-ICN1-MAML complex was formed to activate target genes of Notch1 such as HES (hairy enhancer of split), HRP (HES-related repressor protein), which can precisely regulate cell proliferation and differentiation.In the first part of our experiment, we construct recombined pcDNA3.0-NCT and obtain cell line stably expressing NCT, then cell growth curve and Flow Cytometry was made to analyze cell proliferation and differentiation. But the interesting results showed that NCT overexpression can suppress cell proliferation and improve differentiation. so we assumed that cell proliferation and differentiation can be regulated by a signaling pathway that is associated withγ-secretase.LIF (Leukemia inhibitory factor), was named for its function of inducing terminal differentiation of myeloid leukemia cells and modulating growth/differentiation of many other types of target cells. Additionally, LIF has a lot of other functions including embryogenesis, inflammation and neural development. LIF receptor consists of a low affinity binding protein gp190 and gp130, which is a heterodimer form .Gp190, a type I transmembrane protein, belongs to the hematopoietin receptor family, which is characterized by a cytokine receptor homology (CRH) domain. Cytoplasmic domain of gp190 contains three homologous and functionally important motifs: Box 1, 2 and 3. Gp190 binds to the src-homology 2 (SH2) domain of STAT3 via the YXXQ consensus sequences in the C-terminal. In addition to a membrane form, gp190 also exists in a soluble form that lacks transmembrane domain. Previous reports indicated that certain motif of the cytoplasmic gp190 could activate cell proliferation and differentiation.Considering that gp190 is a type I transmembrane protein, many of which can be cleavaged byγ-secretase, so we supposed thatγ-secretase can cleavage gp190 under physiological condition. In the current study, we constructed recombined plasmid pcDNA3.0-NCT and obtained a cell line of HL-60 stably expressing NCT to enhanceγ-secretase cleavage activity, then cell growth and differentiation was observed. And the second part of the research focus on the study of relationship between LIFR-gp190 andγ-secretase throughγ-secretase inhibitor interference and NCT overexpression which has a role of enhancingγ-secretase cleavage ,the results suggested that overexpression of NCT in HL-60 suppress cell proliferation and induce differentiation. Downregulation of C-terminal fragment of gp190 was observed inγ-secretase inhibitor inference experiment, and more gp190 CTF expression was observed in NCT overexpression HL-60 than pcDNA3.0 transfected HL-60 and wild type HL-60. According to all the results we obtained during our research, we suggested thatγ-secretase can cleavage gp190 under physiological condition and influence signal pathway, the function of which is regulation of cell proliferation and differentiation.PartⅠConstruction of recombined plasmid pcDNA3.0-NCT and expression in HL-60Methods:(1) A set of primer 5'-CGGGGTACCAGAGGCAAGATGGCTACGGCAG-3, 5'-TTTGCGGCCGCCCCAGGGAGGTTCCAGTGACAA-3'was used for amplification of target sequences containing sequence coding for amino acids in protein (CDS) of NCT. Identification of digestion and sequencing was 100% correct; we successfully construct recombined plasmid pcDNA3.0-NCT. (2) We transfected HL-60 with pcDNA3.0-NCT ,G418 was used for a selection of positive clone for 15d , then we obtained a cell line stably expressing NCT, besides, HL-60 transfected with pcDNA3.0 and wild HL-60 was set as control groups. Cells were collected for RT-PCR and western blot analysis of NCT to identify the leukemia cell lines with stable protein expression.(3) Growth curve was used to observe cell growth and CD15/CD11b expression of HL-60 was analyzed by Flow Cytometry.Results: (1) After identification of KpnI and NotI digestion, we observed electrophoretic irradiance strip under the UV light. Sequence detection was consistent with the NCT sequence in Genbank. A 2100bp DNA strip was obtained through PCT reaction. Blast with the original NCT sequence, sequence of obtained production was 100% correct;we successfully reconstructed pcDNA3.0-NCT; (2) Selected by G418 for 15d, cells were centrifugal collected for western blot analysis, and results suggested that HL-60 transfected with pcDNA3.0-NCT expressed more NCT than the other two groups of HL-60, cell line that stably expressing NCT was obtained and preserved for further study; (3)Growth curve showed that proliferation of cells expressing more NCT was obviously suppressed , and CD15/CD11b expression was higher than the other two groups of cells.Conclusion: NCT overexpression can suppress HL-60 proliferation and promote differentiation.PartⅡ: Study of relationship ofγ-secretase and LIFRα-subunit gp190Method: (1)γ-secretase inhibitor interference experiment .After HL-60 cells of all 3 groups were cultured in 12-well plates to a concentration of 1×10~7,γ-secretase inhibitor was added to HL-60 cell culture to suppressγ-secretase activity for 8 hours ,with concentration of 0nM(DMSO),25 nM,50 nM for each plate. Then cells were collected for western blotting analysis of PS1-FL,PS1-NTF,NCT,gp190-FL and gp190-CTF. (2)γ-secretase activity was enhanced by overexpression of NCT, and HL-60 transfected with pcDNA3.0 and wild type HL-60 was set for control groups ,then 3 groups of cells were collected for western blot analysis of PS1-FL,PS1-NTF,NCT,gp190-FL and gp190-CTF.(3)LIF stimulation experient: LIF was added to stimulate gp190 expression, then gp190-FL, gp190-CTF, PS1-FL,PS1-NTF,NCT expression was detected with western blot.(4) STAT3 phosphorylation analysis: pcDNA3.0,pcDNA3.0-NCT,wild type HL-60 with 70% confluent was collected to analyze STAT3 phosphorylation.Results: (1)γ-secretase inhibitor interference experiment showed that expression of gp190-CTF was gradually decreased with the concentration ofγ-secretase inhibitor increases, additionally, downregulation of PS1-NTF and upregulation of PS1-FL was observed during the experiment, which indicates thatγ-secretase activity was suppressed byγ-secretase inhibitor ;(2) HL-60 transfected with pcDNA3.0-NCT expressed more NCT and gp190-CTF than the other two groups of HL-60,besides,with the increase of LIF stimulation time,gp190-CTF expression also gradually increases in every group of HL-60.(3) LIF stimulation experiments showed that with the increase of LIF stimulation time,gp190-CTF was upregulated in three groups of cells. (4) STAT3 phosphorylation analysis suggested that NCT overexpression lead to higher STAT3 phosphorylation level than cells transfected with pcDNA3.0 and wild type HL-60.Conclusion:gp190-CTF expression decreased whenγ-secretase inhibitor was used to suppressγ-secretase activity,and upregulation of gp190-CTF was observed in HL-60 stably expressing more NCT, all of these indicates that gp190 could be cleaved byγ–secretase.SummaryThis research proved that overexpression of NCT can suppress cell proliferation and induce differentiation, which was quite opposite to our expectation that NCT overexpression can enhance cell proliferation through enhancing cleavage of Notch1.Additionally; this research provided conclusive evidence that the C-terminal of the gp190 could be released byγ-secretase cleavage. The released fragment could inhibit the proliferation of leukemia cells; induce their differentiation into granulocytes by enhancing STAT3 phosphorylation and nuclear translocation.