Proinflammatory phenotype and increased caveolin-1 in alveolar macrophages with silenced CFTR mRNA

Abstract

The inflammatory milieu in the respiratory tract in cystic fibrosis (CF) has been linked to the defective expression of the cystic transmembrane regulator (CFTR) in epithelial cells. Alveolar macrophages (AM), important contibutors to inflammatory responses in the lung, also express CFTR. The present study analyzes the phenotype of human AM with silenced CFTR. Expression of CFTR mRNA and the immature form of the CFTR protein decreased 100-fold and 5.2-fold, respectively, in AM transfected with a CFTR specific siRNA (CFTR-siRNA) compared to controls. Reduction of CFTR expression in AM resulted in increased secretion of IL-8, increased phosphorylation of NF-kappaB, a positive regulator of IL-8 expression, and decreased expression of IkappaB-alpha, the inhibitory protein of NF-kappaB activation. AM with silenced CFTR expression also showed increased apoptosis. We hypothesized that caveolin-1 (Cav1), a membrane protein that is co-localized with CFTR in lipid rafts and that is related to inflammation and apoptosis in macrophages, may be affected by decreased CFTR expression. Messenger RNA and protein levels of Cav1 were increased in AM with silenced CFTR. Expression and transcriptional activity of sterol regulatory element binding protein (SREBP), a negative transcriptional regulator of Cav1, was decreased in AM with silenced CFTR, but total and free cholesterol mass did not change. These findings indicate that silencing of CFTR in human AM results in an inflammatory phenotype and apoptosis, which is associated to SREBP-mediated regulation of Cav1.

Figure 1. CFTR knockdown in AM.

Human AM, obtained by bronchoalveolar lavage from healthy adults, were transfected with CFTR-siRNA or control-siRNA and analyzed after 48 h for CFTR mRNA and protein by real-time RT-PCR and Western analysis. To confirm the specificity of CFTR antibody, A549 cells, which do not have intrinsic CFTR expression, and CFTR expressing cell line Calu-3 cells were used as negative and positive controls in the Western analysis. A. Real-time RT-PCR. Human 18s ribosomal RNA was used as normalization control. B. Western analysis. B-tubulin or GAPDH was used as control. CFTR protein was detected as an mmature form (band B) at the size of 150 kDa and a mature form (band C) at the size of 170 kDa. C. and D. Quantification of Western analysis. Shown is the mean ± SEM of three pairs of independent samples. This experiment is the representative of 6 studies.

Figure 2. Increased IL-8 secretion of AM with CFTR knockdown.

A. AM, transfected with CFTR-siRNA or control-siRNA, were analyzed after 6, 12, 18, 24, 36, 48, and 72 h for IL-8 in the cell culture supernatant by ELISA (* denotes p value: p = 0.03 at 24 h; p = 0.01 at 36 h; p = 0.001 at 48 h; p = 0.004 at 72 h). B. AM, transfected with CFTR-siRNA or control-siRNA after 48 h, were evaluated for phosphorylated NF-κB protein expression of by Western analysis. C. Quantification of phosphorylated NF-κB Western analysis. D. AM, transfected with CFTR-siRNA or control-siRNA after 48 h, were also evaluated for IκB-α protein expression of by Western analysis. E. Quantification of IκB-αWestern analysis. Shown is the mean ± SEM of three pairs of independent samples. This experiment is the representative of 6 studies.

Figure 3. Increased apoptosis in AM with deficient CFTR.

AM transfected with CFTR-siRNA or control-siRNA for 48 h were analyzed for apoptosis by TUNEL assay and for cleaved PARP protein expression by Western analysis. A. TUNEL assay. The nuclear staining of green fluorescence was shown as the positive apoptosis signal. DAPI served as normal nuclear staining control. B. Quantification of TUNEL assay. C. IL-8 secretion adjusted to the percentage of non-apoptotic cells in AM transfected with CFTR-siRNA or control-siRNA after 48 h. D. Western analysis of cleaved PARP protein expression using β-tubulin as loading control. E. Quantification of Western analysis. Shown is the mean ± SEM of three pairs of independent samples. This experiment is the representative of 6 studies.

Figure 4. Cav1 expression is increased in AM with deficient CFTR.

AM transfected with CFTR-siRNA or control-siRNA were analyzed for Cav1 mRNA and protein expression after 48 h. A. Real-time RT-PCR. The human 18 s ribosome RNA served as the normalization control. B. Western analysis. B-tubulin was used as control. C. Quantification of Western analysis. Shown is the mean ± SEM of three pairs of independent samples. This experiment is the representative of 6 studies.

Figure 5. Decreased cleavage of the SREBP and transcription activity of SRE in AM with deficient CFTR.

A. AM transfected with CFTR-siRNA or control-siRNA were evaluated after 48 h for SREBP protein expression by Western analysis. B. Quantification of Western analysis. C. Transcription activity assay of SRE. AM, transfected with CFTR-siRNA for 48 h, were infected with AdZ-SRE-luc. The transcriptional activity of SRE was measured by luciferase assay using β-gal as normalization control. D. Total cellular cholesterol, free cholesterol, and cholesterol ester were measured by liquid chromatography. Shown is the mean ± SEM of three pairs of independent samples. This experiment is the representative of 6 studies.