ABCMdb

PMID: 22973054

Veit G, Bossard F, Goepp J, Verkman AS, Galietta LJ, Hanrahan JW, Lukacs GL
Proinflammatory cytokine secretion is suppressed by TMEM16A or CFTR channel activity in human cystic fibrosis bronchial epithelia.
Mol Biol Cell. 2012 Nov;23(21):4188-202. doi: 10.1091/mbc.E12-06-0424. Epub 2012 Sep 12., [PubMed]

Sentences

No. Mutations Sentence Comment

5 ABCC7 p.Gly551Asp Expression of wild-type but not the inactive G551D-CFTR indicates that secretion of the chemoattractant IL-8 is inversely proportional to CFTR channel activity in cftr࢞F508/࢞F508 immortalized and primary human bronchial epithelia. Login to comment 96 ABCC7 p.Gly551Asp After 5 d on filter supports these ratios resulted in a mixed-cell population containing ~10% or ~20% of CFTR-expressing cells, respectively, as determined by immunostaining and cell surface ELISA CFTR channel function is required to suppress IL-8 secretion To assess whether CFTR apical PM expression is involved as a signaling platform (Li and Naren, 2005; Mehta, 2007) or as an anion translocation pathway, we established the effect of the nonfunctional G551D CFTR mutant expression on IL-8 secretion. Login to comment 97 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp G551D and wt CFTR have similar biosynthetic and endocytic processing, but the G551D CFTR cannot be activated by PKA stimulation (Anderson and Welsh, 1992; Barriere et al., 2009). Login to comment 98 ABCC7 p.Gly551Asp The concentration and time dependences of G551D and wt CFTR expression in CFBE cells were similar (Figure 2E and Supplemental Figure S2A). Login to comment 99 ABCC7 p.Gly551Asp As expected, G551D CFTR failed to confer PKA-stimulated PM iodide conductance in the absence of potentiator in both CFBE and H441 epithelia (Supplemental Figure S2, B and C). Login to comment 100 ABCC7 p.Gly551Asp Induction of wt but not G551D CFTR significantly attenuated IL-8 secretion under ALC but not under LLC in both model systems (Figure 2, F and G), strongly suggesting that CFTR channel activity is indispensable for suppressing IL-8 secretion in these polarized respiratory epithelia. Login to comment 105 ABCC7 p.Gly551Asp (D) IB of CFBE or H441 cells transduced with inducible wild-type (iCFTR) or G551D (iG551D) CFTR with (+) or without (-) Dox in comparison to the endogenous CFTR expression in Calu3 cells. Login to comment 106 ABCC7 p.Gly551Asp (E) The dependence of wt and G551D CFTR PM densities on Dox concentration as determined by cell-surface ELISA. Login to comment 107 ABCC7 p.Gly551Asp (F, G) wt or G551D expression-dependent basolateral IL-8 secretion of polarized (F) CFBE or (G) H441 cells switched to ALC or kept under LLC for the indicated times. Login to comment 113 ABCC7 p.Gly551Asp In contrast, G551D CFTR expression had no effect (Figure 4F). Login to comment 129 ABCC7 p.Gly551Asp To confirm the results obtained with inducible epithelial models, we transduced primary human bronchial epithelia (HBE) cells isolated from lung tissue of cftrƊF508/ƊF508 CF individuals (Fulcher et al., 2005) with lentiviral particles (multiplicity of infection [MOI], 4 and 8) conferring constitutive wt or G551D CFTR expression. Login to comment 130 ABCC7 p.Gly551Asp IB and immunostaining indicated that wt or G551D CFTRs were expressed in ~10% of the epithelial cells after 15 d culture on filter supports FIGURE 3:ߒ CFTR function is required to suppress IL-8 secretion. Login to comment 164 ABCC7 p.Gly551Asp (D, E) IB (D) and confocal microscopy pictures (E) of primary CF HBE transduced with lentiviral particles transferring wt, G551D CFTR (green), or empty vector cDNA at a MOI of 4 or 8. Login to comment 187 ABCC8 p.Ile152Leu Consistent results were obtained by measuring the halide conductance with cytosolic YFP-H148Q/I152L/F46L, a halide-sensitive fluorescent protein (Ferrera et al., 2009; Figure 7, E and F, and Supplemental Figure S5I). Login to comment 209 ABCC7 p.Gly551Asp 2) The functionally inactive but normally processed G551D variant cannot substitute for wt CFTR in suppressing IL-8 release. Login to comment 223 ABCC7 p.Gly551Asp IP, immunoprecipitation. but not G551D CFTR expression on IL-8 secretion by primary HBE cells isolated form cftrƊF508/ƊF508 CF individuals. Login to comment 279 ABCC7 p.Gly551Asp Briefly, wt or G551D CFTR harboring an extracellular 3HA tag (Sharma et al., 2004) was subcloned into pLVX-tight-puro using a NotI/blunt-end strategy. Login to comment 313 ABCC7 p.Gly551Asp CFTR cell surface density measurement The PM density of wt and G551D CFTR was determined by cell-surface ELISA (Okiyoneda et al., 2010). Login to comment 321 ABCC7 p.Gly551Asp Passage 1 (P1) or cryopreserved P1 cells were transduced with lentiviral particles mediating cDNA transfer of wt and G551D CFTR harboring an extracellular 3HA tag or empty TranzVector (Tranzyme, Durham, NC; Wu et al., 2000) at MOI of 4 or 8 and subsequently transferred onto collagen IV-coated, 6-mm Transwell filters (Corning, Tewksbury MA) in air-liquid interface medium at a density of 1.2 &#d7; 105 cells/filter. Login to comment 355 ABCC8 p.Ile152Leu Halide permeability determination of the PM The halide sensor YFP-F46L/H148Q/I152L (Namkung et al., 2010) was amplified by PCR and cloned into the XhoI/BamHI restriction sites of the pLVX-IRES-Hyg lentiviral vector (Clontech). Login to comment