Mutations of the ELA2 gene found in patients with severe congenital neutropenia induce the unfolded protein response and cellular apoptosis

Abstract

Severe congenital neutropenia (SCN) is an inborn disorder of granulopoiesis. Mutations of the ELA2 gene encoding neutrophil elastase (NE) are responsible for most cases of SCN and cyclic neutropenia (CN), a related but milder disorder of granulopoiesis. However, the mechanisms by which these mutations disrupt granulopoiesis are unclear. We hypothesize that the ELA2 mutations result in the production of misfolded NE protein, activation of the unfolded protein response (UPR), and ultimately apoptosis of granulocytic precursors. Expression of mutant NE but not wild-type NE strongly induced BiP/GRP78 mRNA expression and XBP1 mRNA splicing, 2 classic markers of the UPR. The magnitude of UPR activation by a specific ELA2 mutation correlated with its associated clinical phenotype. Consistent with the UPR model, expression of mutant NE in primary human granulocytic precursors increased expression of CHOP (DDITS) and induced apoptosis in a protease-independent fashion. Most strikingly, UPR activation and decreased NE protein expression were detected in primary granulocytic precursors from SCN patients. Collectively, these data provide strong support for a UPR model of SCN disease pathogenesis and place SCN in a growing list of human diseases caused by misfolded proteins.

Figure 1

Expression of mutant NE induces XBP1 splicing in U937 cells. U937 cells were transiently transfected with vector alone (GFP), wild-type (WT), or mutant NE cDNAs, and 12 hours later GFP + cells were sorted by flow cytometry. As controls, parent U937 cells (P) and cells treated with tunicamycin (TM) also were analyzed. (A) Representative gel showing spliced (S) and unspliced (US) XBP1 bands. (B) The percentage of spliced XBP1 is shown. Data represent the mean (± SD) of 5 independent experiments. *P < .05 compared with WT-transfected cells.

Figure 2

Expression of mutant NE induces BiP expression in U937 cells. U937 cells were transiently transfected with vector alone (GFP), wild-type (WT), or mutant NE cDNAs, and 12 hours later GFP + cells were sorted by flow cytometry. BiP, NE, and ribosomal RNA (rRNA) were quantified by real-time RT-PCR. (A) Expression of BiP mRNA relative to rRNA is shown. (B) Expression of BiP mRNA to NE mRNA is shown. Data are normalized to WT-transfected cells, where the BiP to NE ratio in each experiment was defined as 1. Data represent the mean (± SD) of 5 independent experiments. *P < .05 compared with WT-transfected cells.

Figure 3

Expression of mutant NE reduces the clonogenic capacity of U937 cells. U937 cells transfected with the indicated NE cDNA were sorted based on GFP expression and plated at limiting dilution in 96-well plates. The number of positive wells on day 14 of culture is shown. Data are normalized to WT-transfected cells, where the number of positive wells was defined as 1. Data represent the mean (± SD) of 3 independent experiments. *P < .05 compared with WT-transfected cells.

Figure 4

Expression of mutant NE activates UPR-induced apoptosis in promyelocytes. Cultured human promyelocytes were transiently transfected with vector alone (GFP) or the indicated NE cDNA. (A) BiP mRNA expression relative to rRNA was determined on sorted GFP⁺ cells 12 hours after transfection. (B,C) Representative scatter plots of annexin V expression on cells transfected with WT (B) or G185R NE (C); data are gated on GFP⁺ 7AAD⁻ cells. Gates were set based on untransfected cells (not shown). (D) The percentage of GFP⁺ 7AAD⁻ cells that were annexin V⁺ at 24 hours after transfection is shown. Data are normalized to WT-transfected cells, where the percentage of annexin V⁺ cells was defined as 1. (E) CHOP mRNA expression relative to rRNA was determined on sorted GFP⁺ cells 12 hours after transfection. Data represent the mean (± SD) of 4 to 6 independent experiments * P < .05 compared with WT-transfected cells.

Figure 5

The protease activity of mutant NE is not required to activate UPR-induced apoptosis. (A) The indicated NE cDNA was transiently transfected into RBL cells, and 24 hours later cell extracts were prepared from bulk cultures. NE protease activity was quantified using a chromogenic, NE-sensitive substrate. Shown is NE protease activity after correcting for the number of GFP⁺ cells analyzed. (B,C) Cultured human granulocytic precursors were transiently transfected with the indicated NE cDNA. (B) BiP mRNA expression relative to rRNA was determined on sorted GFP⁺ cells 12 hours after transfection. (C) The percentage of GFP⁺ 7AAD⁻ cells that were annexin V⁺ at 24 hours after transfection is shown. Data represent the mean (± SD) of 4-5 independent experiments. *P < .05 compared with WT-transfected cells. NS indicates nonsignificant.

Figure 6

The UPR is activated in SCN promyelocytes/granulocytes. (A) Representative scatter plots of bone marrow cells from a healthy donor (normal) and a patient with ELA2-mutation positive SCN (A28T) stained for CD15 and CD16; data are gated to exclude CD14⁺ monocytes and CD9⁺ eosinophils. The boxed region shows the sorted CD15⁺ CD16^lo CD14⁻ CD9⁻ cell population. (B) The percentage of spliced XBP-1 mRNA in the sorted cell population is shown. (C) Expression of BiP mRNA relative to NE mRNA in the sorted cell population is shown.

Figure 7

NE and MPO protein expression is decreased in SCN granulocytic precursors. Confocal microscopy of 2 different healthy donors (normal) and 3 ELA2-mutant SCN samples stained with NE and myeloperoxidase (MPO; a primary granule constituent). Isotype controls for NE and MPO are shown. Data are representative of 6 normal and 6 ELA2 mutation-positive SCN bone marrow samples.