In vivo alterations of IFN regulatory factor-1 and PIAS1 protein levels in cystic fibrosis epithelium (original) (raw)
IRF-1 is a necessary factor for constitutive epithelial NOS2 expression. It has already been shown that inducible expression of NOS2 is diminished in macrophages and glial cells isolated from mice lacking IRF-1 expression (IRF-1–/– mice) (14, 15). We examined NOS2 expression in excised nasal epithelium from C57BL/6J mice and IRF-1–/– mice with the same C57BL/6J background to determine if IRF-1 expression is necessary for constitutive epithelial NOS2 production. We have shown previously that NOS2–/– mice with a C57BL/6J background lack NOS2 staining, demonstrating the lack of nonspecific binding of the NOS2 antibody in mice with the C57BL/6J background (3). NOS2-specific immunostaining revealed that normal C57BL/6J mice express detectable NOS2 in nasal epithelium, but that this NOS2 expression is reduced in IRF-1–/– mice (Figure 2). The attenuated level of NOS2 expression in the nasal epithelium of IRF-1–/– mice demonstrates dependence on IRF-1 for constitutive NOS2 expression, and its expression should be explored in CF epithelium.
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).
IRF-1–mediated regulation of transepithelial chloride transport. To establish a functional effect of reduced IRF-1 expression on epithelial NO production, we examined the chloride-free response using the nasal TEPD assay in IRF-1–/– mice and normal C57BL/6J mice. The chloride-free response was measured by perfusing the nasal passage of the mice with a chloride-free Ringer’s solution in the presence of the sodium-transport inhibitor amiloride. Absence of chloride in the Ringer’s solution establishes a driving force for the secretion of chloride across the nasal epithelium into the lumen. This response can be considered a measurement of basal chloride transport properties of the epithelium in that no chloride secretory agonists are present. We have shown previously that NOS2–/– mice have a significantly reduced chloride-free response compared with normal C57BL/6J mice, but this response returns to near wild-type levels when the NO donor sodium nitroprusside (SNP) is added to the perfusion solution (4). These data point to a role for NO in regulating basal transepithelial chloride transport. This experiment was repeated using IRF-1–/– mice. Wild-type C57BL/6J mice show a –9.9 ± 1.2 mV (n = 6) hyperpolarization in response to chloride-free Ringer’s after 3 minutes, indicating robust chloride secretion across the nasal epithelium. IRF-1–/– mice, however, showed a hyperpolarization of only –2.1 ± 1.1 mV (n = 8) under the same conditions, indicating reduced endogenous stimulus for chloride transport that was essentially identical to that observed with the NOS2 mice (Figure 3) (4). When the NO donor SNP (100 μM) is added to the perfusion solution, the chloride secretory response increases to a –8.5 ± 1.0 mV (n = 5) hyperpolarization in IRF-1–/– mice. These data show a functional consequence of attenuated NO production due to the elimination of IRF-1 expression. Consistent with previous findings, IRF-1 is a factor essential for the proper regulation of epithelial NO production in vivo.
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.
In vivo constitutive IRF-1 expression in CF and non-CF epithelium. Examination of IRF-1 expression levels from excised nasal epithelium from CFTR+/– mice and CFTR–/– mice revealed that IRF-1 levels were significantly reduced in CFTR–/– nasal epithelium compared with those in epithelium from wild-type heterozygous mice (Figure 4a). However, a high-molecular-weight band that was recognized by the IRF-1 antibody did appear in some, but not all, of the CFTR–/– nasal epithelial samples (not shown). The presence of this band in only some preparations of nasal epithelium inclines us to believe it is a contaminant, but the significance of this high-molecular-weight immunoreactive band in the CF nasal epithelial samples will have to be determined. The figure presents results from three separate CFTR+/– mice and three separate CFTR–/– mice. Densitometry results are representative of eight wild-type mice and seven CF mice tested. IRF-1 expression is significantly lower in CFTR–/– mice, with a P value of 0.002.
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-h_cftr_ 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.
We have shown that constitutive NOS2 expression is dependent on the presence of functional CFTR in epithelial cells from excised ileum and nasal tissues (26). To determine whether IRF-1 and NOS2 expression are influenced by similar CFTR-dependent regulatory mechanisms, we examined IRF-1 expression in the same mouse models used previously to study NOS2 expression. Three groups of mice were examined for IRF-1 expression in epithelial cells excised from the ileum. These groups consisted of CFTR+/– mice, CFTR–/– mice, and additional CFTR–/– mice that express human CFTR specifically in intestinal epithelium driven by the fatty-acid binding protein (FABP) promoter (FABP-h_cftr_ mice) (26, 27). IRF-1 is constitutively expressed in the ileum of CFTR+/– mice and FABP-h_cftr_ mice, but is not expressed in tissue isolated from CFTR–/– mice (Figure 4c). These data demonstrate the same CFTR-dependent IRF-1 expression that we demonstrated previously in the case of NOS2 expression (26). The reintroduction of functional CFTR into the mouse intestinal epithelium restores the expression of IRF-1. Densitometric analysis of protein expression reveals no significant difference in IRF-1 expression between wild-type and FABP-h_cftr_ mouse ileum, but shows a significant reduction in the ileum of CFTR–/– mice.
Stat1 and PIAS1 expression in CF and non-CF murine epithelium. Our data indicate that expression of NOS2 and IRF-1 are diminished in CF epithelial cells by a mechanism related to diminished CFTR expression or function. Because NOS2 and IRF-1 expression are dependent on the activation of Stat1, we examined expression levels of Stat1 in CF and non-CF mouse nasal epithelium. Stat1 protein levels were found to be overexpressed in excised nasal epithelium from CFTR–/– mice compared with CFTR+/+ mice (Figure 5a), which is surprising in light of the apparent reduction of IRF-1 expression in CF epithelium.
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.
The apparent reduction of IRF-1 and NOS2 expression suggests that there is an interruption in normal Stat1 signaling. Because Stat1 levels are increased, we also examined expression levels of the known Stat1 inhibitor PIAS1. PIAS1, also identified as Gu RNA helicase-II binding protein (GBP) (28), has been shown to bind to Stat1 and to specifically inhibit Stat1-mediated signaling events (29). PIAS1 levels were elevated in excised nasal epithelium from CFTR–/– mice, but were barely detectable in CFTR+/– and CFTR+/+ mice (Figure 5c). We have shown previously that NO production and NOS2 mRNA expression is reduced in a 9/HTEo– cell model of CF (26). Mock-transfected 9/HTEo– cells (pCEP2) serve as wild-type cells, whereas cells transfected to overexpress the CFTR R domain (pCEPRF) lack cAMP-mediated chloride secretion and have several CF-like phenotypes (7, 25). PIAS1 levels were found to be significantly higher in the CF-phenotype cells (pCEPRF) than in the wild-type phenotype cells (pCEP2) (Figure 5e). Differences in protein expression were quantified by densitometry. Significantly higher expression of both Stat1 and PIAS1 proteins was found in CF samples (Figure 5, b, d, and f). Increased expression of PIAS1 raises the possibility that Stat1 signaling may be less than optimal in CF epithelium despite the presence of increased levels of Stat1 protein.
PIAS1-bound Stat1 in CF and non-CF mouse nasal epithelium. To address functional differences associated with increased PIAS1 levels in CF epithelium, we examined levels of free and PIAS1-bound Stat1. Stat1 is activated by phosphorylation via the Janus kinase (Jak) family, leading to Stat1 nuclear translocation, dimerization, and DNA binding. PIAS1 binds to phosphorylated Stat1, preventing dimerization and DNA binding (29). We assessed differences in anti-phosphotyrosine-accessible Stat1, and Stat1 that is apparently associated with PIAS1 in CF and non-CF cells. Immunoprecipitation with anti-phosphotyrosine antibodies revealed a lack of p-Stat1 in samples of nasal epithelium from CFTR–/– mice, whereas Stat1 levels were clearly detectable in CFTR+/– mouse nasal tissue (Figure 6a). Although PIAS1 is not reported to bind directly to the phosphorylated site of Stat1, we postulated that perhaps PIAS1 binding to p-Stat1 could interfere with anti-phosphotyrosine antibody binding to p-Stat1. We therefore immunoprecipitated with anti-PIAS1 and examined precipitated samples for associated Stat1 to determine if p-Stat1 was interacting with increased levels of PIAS1 in CF epithelial cells. Although more PIAS1 was isolated in CF samples by immunoprecipitation with anti-PIAS1 as expected, similar levels of Stat1 were found in samples from both CFTR+/– and CFTR–/– mouse nasal epithelium (Figure 6b). To determine if Stat1 bound to PIAS1 was indeed phosphorylated, we performed the same experiment of immunoprecipitating PIAS1 and probing the blot with an antibody specific for the phosphorylated form of Stat1. Both CF and non-CF mouse nasal epithelial samples and the 9/HTEo– pCEP2 and pCEPRF cells were tested (Figure 7, a and b, respectively). Phosphorylated Stat1 was bound to PIAS1 in all of the samples. These data demonstrate that p-Stat1 is present in CF cells, but the majority of available active p-Stat1 is apparently bound to PIAS1. This observation is true both in vivo in mouse CF epithelial cells and in the CF cell model 9/HTEo– pCEP2 and pCEPRF cells. Increased PIAS1 levels may interfere with full cellular Stat1 signaling.
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.
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.
Stat1-mediated signaling in 9/HTEo– pCEP2 and pCEPRF cells. The CF-phenotype 9/HTEo– pCEPRF cells reflected the same decrease of NOS2 expression (26), the same increase of PIAS1 protein expression (Figure 5), and the same PIAS1-mediated binding of p-Stat1 (Figure 6) seen in CF mouse nasal epithelium, compared with respective wild-type controls. Therefore, the ability of these cells to carry out Stat1-mediated signaling was tested. Activated Stat1 specifically binds to the IFN-γ–activated site (GAS) to mediate gene transcription, so the luciferase reporter construct pGAS-Luc was transfected into control pCEP2 and CF-phenotype pCEPRF cells. Cells were treated with 0, 10, 25, or 100 units/mL of recombinant human IFN-γ for 5 hours in serum-free media (Figure 8). The CF-phenotype pCEPRF cells had significantly lower basal luciferase activity and significantly lower luciferase activity at each tested concentration of IFN-γ than was observed in the control pCEP2 cells. IFN-γ also significantly stimulated luciferase activity at each concentration tested in pCEP2 cells, whereas only the two higher concentrations of IFN-γ had a measurable effect in the pCEPRF cells. The effect of serum on stimulated luciferase activity was also tested in pCEP2 and pCEPRF cells. Significant stimulation of luciferase activity was observed in the control 9/HTEo– pCEP2 cells, but no stimulation occurred in the CF-phenotype pCEPRF cells. Data were normalized for transfection efficiency by measuring GFP fluorescence mediated by cotransfection with pEGFPN1 as described in Methods, and are presented as a ratio of RLU to RFU. These data demonstrate that there is an inherent decrease in Stat1-mediated signaling in the CF-phenotype 9/HTEo– pCEPRF cells that is consistent with an increase in the expression of PIAS1 protein.
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. A_P_ < 0.001; B_P_ < 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 (C_P_ < 0.01; D_P_ < 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 (E_P_ < 0.01; F_P_ < 0.0001).