Decreased human epididymis protein 4 (HE4) plasma levels were reported in cystic fibrosis (CF) patients under CFTR potentiator ivacaftor therapy, which inversely correlated with lung function improvement. In this study, we investigated whether HE4 expression was affected via modulation of CFTR function in CF bronchial epithelial (CFBE) cells in vitro. HE4 protein levels were measured in the supernatants of CFBE 41o- cells expressing F508del-CFTR or wild-type CFTR (wt-CFTR) after administration of lumacaftor/ivacaftor or tezacaftor/ivacaftor, while HE4 expression in CFBE 41o- cells were also analyzed following application of adenylate cyclase activators Forskolin/IBMX or CFTRinh172. The effect of all of these compounds on CFTR function was monitored by the whole-cell patch-clamp technique. Induced HE4 expression was studied with interleukin-6 (IL-6) in F508del-CFTR CFBE 41o- cells under TNF-α stimulation for 1 h up to 1 week in duration. In parallel, plasma HE4 was determined in CF subjects homozygous for p.Phe508del-CFTR mutation receiving lumacaftor/ivacaftor (Orkambi®) therapy. NF-κB-mediated signaling was observed via the nuclear translocation of p65 subunit by fluorescence microscopy together with the analysis of IL-6 expression by an immunoassay. In addition, HE4 expression was examined after NF-κB pathway inhibitor BAY 11-7082 treatment with or without CFTR modulators. CFTR modulators partially restored the activity of F508del-CFTR and reduced HE4 concentration was found in F508del-CFTR CFBE 41o- cells that was close to what we observed in CFBE 41o- cells with wt-CFTR. These data were in agreement with decreased plasma HE4 concentrations in CF patients treated with Orkambi®. Furthermore, CFTR inhibitor induced elevated HE4 levels, while CFTR activator Forskolin/IBMX downregulated HE4 in the cell cultures and these effects were more pronounced in the presence of CFTR modulators. Higher activation level of baseline and TNF-α stimulated NF-κB pathway was detected in F508del-CFTR vs. wt-CFTR CFBE 41o- cells that was substantially reduced by CFTR modulators based on lower p65 nuclear positivity and IL-6 levels. Finally, HE4 expression was upregulated by TNF-α with elevated IL-6, and both protein levels were suppressed by combined administration of NF-κB pathway inhibitor and CFTR modulators in CFBE 41o- cells. In conclusion, CFTR dysfunction contributes to abnormal HE4 expression via NF-κB in CF.
- Publikační typ
- časopisecké články MeSH
Cystic fibrosis (CF) is a monogenetic disease resulting from mutations in the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) gene encoding an anion channel. Recent evidence indicates that CFTR plays a role in other cellular processes, namely in development, cellular differentiation and wound healing. Accordingly, CFTR has been proposed to function as a tumour suppressor in a wide range of cancers. Along these lines, CF was recently suggested to be associated with epithelial-mesenchymal transition (EMT), a latent developmental process, which can be re-activated in fibrosis and cancer. However, it is unknown whether EMT is indeed active in CF and if EMT is triggered by dysfunctional CFTR itself or a consequence of secondary complications of CF. In this study, we investigated the occurrence of EMT in airways native tissue, primary cells and cell lines expressing mutant CFTR through the expression of epithelial and mesenchymal markers as well as EMT-associated transcription factors. Transepithelial electrical resistance, proliferation and regeneration rates, and cell resistance to TGF-β1induced EMT were also measured. CF tissues/cells expressing mutant CFTR displayed several signs of active EMT, namely: destructured epithelial proteins, defective cell junctions, increased levels of mesenchymal markers and EMT-associated transcription factors, hyper-proliferation and impaired wound healing. Importantly, we found evidence that the mutant CFTR triggered EMT was mediated by EMT-associated transcription factor TWIST1. Further, our data show that CF cells are over-sensitive to EMT but the CF EMT phenotype can be reversed by CFTR modulator drugs. Altogether, these results identify for the first time that EMT is intrinsically triggered by the absence of functional CFTR through a TWIST1 dependent mechanism and indicate that CFTR plays a direct role in EMT protection. This mechanistic link is a plausible explanation for the high incidence of fibrosis and cancer in CF, as well as for the role of CFTR as tumour suppressor protein.
- MeSH
- cystická fibróza metabolismus patologie MeSH
- epitelo-mezenchymální tranzice MeSH
- HEK293 buňky MeSH
- jaderné proteiny metabolismus MeSH
- kultivované buňky MeSH
- lidé MeSH
- onkogeny genetika MeSH
- protein CFTR metabolismus MeSH
- transkripční faktor Twist metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
BACKGROUND: For most of the >2000 CFTR gene variants reported, neither the associated disease liability nor the underlying basic defect are known, and yet these are essential for disease prognosis and CFTR-based therapeutics. Here we aimed to characterize two ultra-rare mutations - 1717-2A > G (c.1585-2A > G) and S955P (p.Ser955Pro) - as case studies for personalized medicine. METHODS: Patient-derived rectal biopsies and intestinal organoids from two individuals with each of these mutations and F508del (p.Phe508del) in the other allele were used to assess CFTR function, response to modulators and RNA splicing pattern. In parallel, we used cellular models to further characterize S955P independently of F508del and to assess its response to CFTR modulators. RESULTS: Results in both rectal biopsies and intestinal organoids from both patients evidence residual CFTR function. Further characterization shows that 1717-2A > G leads to alternative splicing generating <1% normal CFTR mRNA and that S955P affects CFTR gating. Finally, studies in organoids predict that both patients are responders to VX-770 alone and even more to VX-770 combined with VX-809 or VX-661, although to different levels. CONCLUSION: This study demonstrates the high potential of personalized medicine through theranostics to extend the label of approved drugs to patients with rare mutations.
- MeSH
- alely MeSH
- aminofenoly terapeutické užití MeSH
- aminopyridiny terapeutické užití MeSH
- benzodioxoly terapeutické užití MeSH
- chinolony terapeutické užití MeSH
- cystická fibróza farmakoterapie genetika MeSH
- elektrofyziologie MeSH
- fluorescenční protilátková technika MeSH
- genotyp MeSH
- individualizovaná medicína metody MeSH
- indoly terapeutické užití MeSH
- lidé MeSH
- mutace genetika MeSH
- protein CFTR genetika metabolismus MeSH
- western blotting MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
BACKGROUND: Increased expression of the human epididymis protein 4 (HE4) was previously described in lung biopsy samples from patients with cystic fibrosis (CF). It remains unknown, however, whether serum HE4 concentrations are elevated in CF. METHODS: Seventy-seven children with CF from six Hungarian CF centers and 57 adult patients with CF from a Czech center were enrolled. In addition, 94 individuals with non-CF lung diseases and 117 normal control subjects with no pulmonary disorders were analyzed. Serum HE4 levels were measured by using an immunoassay, and their expression was further investigated via the quantification of HE4 messenger RNA by using quantitative reverse transcription polymerase chain reaction in CF vs non-CF respiratory epithelium biopsy specimens. The expression of the potential regulator miR-140-5p was analyzed by using an UPL-based quantitative reverse transcription polymerase chain reaction assay. HE4 was measured in the supernatants from unpolarized and polarized cystic fibrosis bronchial epithelial cells expressing wild-type or F508del-CFTR. RESULTS: Median serum HE4 levels were significantly elevated in children with CF (99.5 [73.1-128.9] pmol/L) compared with control subjects (36.3 [31.1-43.4] pmol/L; P < .0001). This observation was replicated in adults with CF (115.7 [77.8-148.7] pmol/L; P < .0001). In contrast, abnormal but lower HE4 concentrations were found in cases of severe bronchitis, asthma, pneumonia, and bronchiectasis. In patients with CF, the concentrations of HE4 were positively correlated with overall disease severity and C-reactive protein concentrations, whereas a significant inverse relationship was found between HE4 and the spirometric FEV1 value. Relative HE4 mRNA levels were significantly upregulated (P = .011) with a decreased miR-140-5p expression (P = .020) in the CF vs non-CF airway biopsy specimens. Twofold higher HE4 concentrations were recorded in the supernatant of polarized F508del-CF transmembrane conductance regulator/bronchial epithelial cells compared with wild-type cells. CONCLUSIONS: HE4 serum levels positively correlate with the overall severity of CF and the degree of pulmonary dysfunction. HE4 may thus be used as a novel inflammatory biomarker and possibly also as a measure of treatment efficacy in CF lung disease.
- MeSH
- bronchiální astma genetika metabolismus MeSH
- bronchiektazie genetika metabolismus MeSH
- bronchitida genetika metabolismus MeSH
- C-reaktivní protein metabolismus MeSH
- cystická fibróza genetika metabolismus patofyziologie MeSH
- dítě MeSH
- dospělí MeSH
- epitelové buňky metabolismus MeSH
- lidé MeSH
- messenger RNA metabolismus MeSH
- mikro RNA metabolismus MeSH
- mladiství MeSH
- mladý dospělý MeSH
- pneumonie genetika metabolismus MeSH
- polymerázová řetězová reakce s reverzní transkripcí MeSH
- předškolní dítě MeSH
- protein CFTR genetika MeSH
- proteiny genetika metabolismus MeSH
- respirační sliznice metabolismus MeSH
- spirometrie MeSH
- studie případů a kontrol MeSH
- stupeň závažnosti nemoci MeSH
- usilovný výdechový objem MeSH
- Check Tag
- dítě MeSH
- dospělí MeSH
- lidé MeSH
- mladiství MeSH
- mladý dospělý MeSH
- mužské pohlaví MeSH
- předškolní dítě MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
