In vivo alterations of IFN regulatory factor-1 and PIAS1 protein levels in cystic fibrosis epithelium

Abstract

Inducible nitric oxide synthase-2 (NOS2) expression has been shown to be reduced in cystic fibrosis (CF) epithelial cells. Reduced NOS2 expression is unexpected, given the inflammatory nature of CF airway disease, and is an indication that cell-signaling mechanisms necessary for proper NOS2 regulation are probably altered in CF epithelium. Therefore, we examined the expression levels of regulatory factors necessary for NOS2 expression in CF epithelium and showed that IFN regulatory factor-1 (IRF-1) is necessary for full NOS2 expression. Mice lacking IRF-1 expression have diminished epithelial NOS2 expression, as well as reduced NO-dependent chloride transport across the nasal epithelia. Furthermore, IRF-1 protein expression is reduced in nasal and intestinal epithelial cells from CF mice, suggesting a possible mechanism for the CF-related reduction of epithelial NOS2 expression. Active signal transducer and activator of transcription-1 (Stat1) is necessary for both NOS2 and IRF-1 expression. We found that protein levels of Stat1 were increased in CF cells, but that the active phosphorylated form of Stat1 was bound to the protein inhibitor of activated Stat1 (PIAS1). We propose that increased levels of PIAS1 diminish certain cell-signaling pathways, resulting in reduced IRF-1 and NOS2 expression in CF epithelial cells.

Figure 1

Cell-signaling components involved in the regulation of NOS2 expression.

Figure 2

NOS2-specific immunostaining in excised murine nasal epithelial tissue. Bright-field view of nasal epithelial tissue from wild-type C57BL/6J mice (a) and IRF-1^–/– mice (b). NOS2-specific fluorescent immunostaining from wild-type C57BL/6J mice (c) and IRF-1^–/– mice (d). IRF-1^–/– mice have a C57BL/6J background. Images are representative of results obtained with four mice of each genotype. Samples processed identically but not incubated with anti-NOS2 antibody showed no staining (data not shown). Objective images (×40).

Figure 3

Murine nasal transepithelial chloride transport is partially IRF-1 dependent. Changes in TEPD in response to perfusion with chloride-free Ringer’s solution were measured in wild-type C57BL/6J mice (filled squares; n = 6), IRF-1^–/– mice (open circles; n = 8), and IRF-1^–/– mice with SNP (100 μM) added to the perfusion solution (filled triangles; n = 5). Error bars represent SEM. Time zero refers to the point at which the perfusion solution was changed to chloride-free Ringer’s (this was done when a plateau value was reached in chloride-replete Ringer’s containing amiloride). Amiloride (100 μM) was present in all perfusion solutions.

Figure 4

IRF-1 expression in CF and non-CF epithelial tissue. (a) Representative blot of IRF-1 protein expression in excised nasal epithelium from CFTR^+/– mice (lanes 1–3) and CFTR^–/– mice (lanes 4–6). Each lane represents tissue isolated from individual mice. (c) IRF-1 expression in excised epithelial tissue from sections of ileum isolated from a CFTR^+/– mouse (lane 1), an FABP-hcftr mouse (lane 2), and a CFTR^–/– mouse (lane 3). (b and d) Densitometric analysis of IRF-1–specific immunoblots. Number of samples (n) is shown in parentheses above each bar. Significance determined by Student’s t test. Error bars represent SEM. NS, no significant difference between these two values. IB, immunoblot.

Figure 5

Stat1 and PIAS1 expression in CF and non-CF model systems. (a) Representative blot of Stat1 protein expression in excised nasal epithelium from CFTR^+/+ mice (lanes 1 and 2) and CFTR^–/– mice (lanes 3 and 4). Each lane represents tissue isolated from individual mice. (c) PIAS1 expression in excised nasal epithelial tissue from CFTR^+/– mice (lanes 1–4) and CFTR^–/– mice (lanes 5–8). This blot was reprobed for erk as a control for protein loading. (e) PIAS1 expression in 9/HTEo^– pCEP2 control cells (lanes 1 and 2) and CF-phenotype 9/HTEo^– pCEPRF cells (lanes 3 and 4). (b, d, f) Densitometric analysis of Stat1, PIAS1, and erk expression. Number of samples (n) is shown in parentheses above each bar. Significance determined by Student’s t test. Error bars represent SEM.

Figure 6

Levels of free and PIAS1-bound Stat1 in CF and non-CF mouse nasal epithelial tissue. (a) Upper panel: Representative blot of Stat1 protein expression in excised nasal epithelium from CFTR^+/– mice (lanes 1–4) and CFTR^–/– mice (lanes 5–7). Lower panel: Free p-Stat1 levels in the same samples as determined by immunoprecipitation with anti-phosphotyrosine and probing with anti-Stat1. Each lane represents tissue isolated from individual mice. (b) Upper panel: Levels of PIAS1 in excised nasal epithelium from CFTR^+/– mice (lanes 1–4) and CFTR^–/– mice (lanes 5–8) as determined by immunoprecipitation with anti-PIAS1 and probing with the same antibody. Lower panel: The same immunoprecipitates shown in the upper panel, probed for Stat1 levels. IP, immunoprecipitate; IB, immunoblot.

Figure 7

Levels of p-Stat1 coprecipitated with PIAS1. To determine if Stat1 bound to PIAS1 was phosphorylated, PIAS1 was immunoprecipitated from nasal epithelial extracts from four CFTR^+/+ and three CFTR^–/– mice (a), and from four different preparations of 9/HTEo^– pCEP2 and pCEPRF cells (b). Precipitates were blotted and probed for immunoreactivity with both anti-PIAS1 and anti–p-Stat1 antibodies.

Figure 8

Stat1-mediated cell signaling in 9/HTEo^– pCEP2 and pCEPRF cells. Cells were cotransfected with pGAS-Luc and pEGFPN1 and stimulated with either 10, 25, or 100 units/mL IFN-γ or with 10% serum as described. Data were normalized for transfection efficiency as measured by GFP expression, and are presented as a ratio of relative light units (RLU) to relative fluorescence units (RFU). The number of experiments (n) is shown in parentheses above each bar. Error bars represent SEM. ^AP < 0.001; ^BP < 0.0001, pCEP2 vs. pCEPRF cells under each experimental condition, by Student’s t test. ^C,DSignificant stimulation of luciferase activity in pCEP2 cells compared with untreated (NT) pCEP2 cells as measured by Duncan’s multiple range test (^CP < 0.01; ^DP < 0.0001). ^E,FSignificant stimulation of luciferase activity in pCEPRF cells compared with untreated (NT) pCEPRF cells, as measured by Duncan’s multiple range test (^EP < 0.01; ^FP < 0.0001).