A Truncated Granulocyte Colony-stimulating Factor Receptor (G-CSFR) Inhibits Apoptosis Induced by Neutrophil Elastase G185R Mutant: IMPLICATION FOR UNDERSTANDING CSF3R GENE MUTATIONS IN SEVERE CONGENITAL NEUTROPENIA

Abstract

Mutations in ELANE encoding neutrophil elastase (NE) have been identified in the majority of patients with severe congenital neutropenia (SCN). The NE mutants have been shown to activate unfolded protein response and induce premature apoptosis in myeloid cells. Patients with SCN are predisposed to acute myeloid leukemia (AML), and progression from SCN to AML is accompanied by mutations in CSF3R encoding the granulocyte colony-stimulating factor receptor (G-CSFR) in ∼80% of patients. The mutations result in the expression of C-terminally truncated G-CSFRs that promote strong cell proliferation and survival. It is unknown why the CSF3R mutations, which are rare in de novo AML, are so prevalent in SCN/AML. We show here that a G-CSFR mutant, d715, derived from an SCN patient inhibited G-CSF-induced expression of NE in a dominant negative manner. Furthermore, G-CSFR d715 suppressed unfolded protein response and apoptosis induced by an SCN-derived NE mutant, which was associated with sustained activation of AKT and STAT5, and augmented expression of BCL-XL. Thus, the truncated G-CSFRs associated with SCN/AML may protect myeloid precursor cells from apoptosis induced by the NE mutants. We propose that acquisition of CSF3R mutations may represent a mechanism by which myeloid precursor cells carrying the ELANE mutations evade the proapoptotic activity of the NE mutants in SCN patients.

Keywords: apoptosis; cell differentiation; cell surface receptor; leukemia; neutrophil.

FIGURE 1.

Expression of NE G185R induces accelerated apoptosis in 32D/GR cells. A, 32D/GR cells were transfected with the empty pBabe-puro vector (32D/Ctr) or expression constructs for NE (32D/NE) or NE G185R (32D/G185R) and examined for expression of NE proteins by Western blotting. B and C, 32D/GR clones as indicated were cultured in G-CSF. The numbers of viable cells and cell viability were determined on different days by exclusion of trypan blue staining. Error bars represent S.D. D, cells as indicated were cultured in G-CSF for 4 days and stained with annexin V and 7-aminoactinomycin (7AAD). The percentages of annexin V-positive (early and late apoptotic) cells are indicated. E, morphological features of 32D/Ctr, 32D/NE, and 32D/G185R cells following culture in G-CSF for 5 days. F, 32D/NE and 32D/G185R cells were cultured in G-CSF for 5 days prior to evaluation of surface expression of Gr-1 by flow cytometry. ** denotes p < 0.01.

FIGURE 2.

Subcellular localization of NE and NE G185R in 32D/GR cells. A, NE proteins in 32D/Ctr, 32D/NE, and 32D/G185R cells were examined by immunofluorescence staining using the monoclonal NE antibody. Essentially identical staining patterns were seen using the polyclonal anti-NE antibody. B, membrane (mem) and cytosolic (cyto) extracts were prepared from cells as indicated and examined for NE proteins by Western blotting. Aliquots of extracts were also examined for β-actin and G-CSFR.

FIGURE 3.

The C-terminal region of G-CSFR is required for induction of NE expression. A, schematic diagram of WT and d715 forms of G-CSFR. Boxes 1, 2, and 3 denote cytoplasmic regions conserved among the members of the cytokine receptor superfamily. TM, transmembrane domain; aa, amino acids. B, cells as indicated were cultured in G-CSF for different days. Total RNA was extracted and examined for NE transcript by Northern blotting analysis. Sample loadings were determined by ethidium bromide staining of 18S ribosomal RNA. C, differential activation of the Elane and IRF-1 promoters by the WT and d715 forms of G-CSFR. 32D/GR and 32D/d715 cells were transfected with the Elane or IRF-1 promoter-luciferase reporter construct along with the pCMV-β-gal plasmid. Cells were left untreated or treated with G-CSF for 6 h. Luciferase activity was measured and normalized for β-galactosidase activity. Error bars represent S.D. ** denotes p < 0.01.

FIGURE 4.

The effect of NE G185R expression driven by Elane promoter on G-CSF-dependent proliferation and survival. A, 32D/GR and 32D/d715 cells were transfected with pcDNA3.1-derived expression construct for NE G185R in which the expressed of NE G185R was under the control of the Elane promoter (ElaP) or the pBabe-puro-derived NE G185R expression vector. The expression of NE G185R in cells cultured in IL-3 was examined by Western blotting analysis. B, 32D/GR and 32D/d715 cells expressing NE G185R from ElaP were cultured in G-CSF and examined for expression of NE G185R. C and D, 32D/GR and 32D/d715 cells transfected with the empty vector (Ctr) or the ElaP-driven NE G185R were cultured in G-CSF. Cell numbers and viabilities were determined on different days. Error bars represent S.D. ** denotes p < 0.01.

FIGURE 5.

G-CSFR d715 suppresses apoptosis induced by NE G185R in 32D cells. 32D/d715 cells were transfected with the pBabe-puro-derived NE G185R expression construct. A, the expression of NE G185R protein was examined by Western blotting analysis. B, two independent 32D/d715 clones expressing NE G185R were cultured in G-CSF, and the numbers of viable cells were determined on different days. C, surface expression of Gr-1 in one 32D/d715 clone was examined by flow cytometry after culture in G-CSF for 5 days.

FIGURE 6.

G-CSFR d715 blocks apoptosis induced by NE G185R in FDCP-mix A4 cells. A, surface expression of human WT G-CSFR (GR) or G-CSFR d715 was examined by flow cytometry. B, the expression of NE G185R in FDCP/GR and FDCP/d715 cells was examined by Western blotting analysis. Cells were subsequently cultured in G-CSF, and the numbers of viable cells (C) and cell viabilities (D) were determined daily. Error bars represent S.D. E, cells as indicated were cultured in G-CSF for 4 days. Apoptotic cell death was evaluated by annexin V assay. Numbers indicate the percentages of annexin V-positive cells. ** denotes p < 0.01. 7AAD, 7-aminoactinomycin.

FIGURE 7.

G-CSFR d715 suppresses Grp78 induction by NE G185R. Control (Ctr) and NE G185R-expressing 32D and FDCP-mix A4 cells transfected with the WT or d715 form of G-CSFR were cultured in G-CSF for 24 h. The expression of murine Grp78 mRNA was assessed by qRT-PCR. Error bars represent S.D. * denotes p < 0.05; ** denotes p < 0.01.

FIGURE 8.

G-CSF-stimulated activation of STAT5 and AKT in NE G185R-expressing 32D and FDCP-mix A4 cells transfected with the WT or d715 form of G-CSFR. A, cells as indicated were starved of IL-3 for 2 h prior to stimulation with G-CSF for the indicated minutes. B, cells were cultured in IL-3 (day 0) or G-CSF for up to 3 days. The phosphorylation (p) and expression of STAT5 and AKT were examined by Western blotting analysis.

FIGURE 9.

Expression of BCL-XL, BCL-2, and MCL-1 in NE G185R-expressing 32D and FDCP-mix A4 cells transfected with the WT or d715 form of G-CSFR. Cells as indicated were cultured in IL-3 (day 0) or G-CSF for up to 4 days. The expression of BCL-XL, BCL-2, and MCL-1 was examined by Western blotting analysis.