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1.  Functional Stability of Rescued ΔF508 Cystic Fibrosis Transmembrane Conductance Regulator in Airway Epithelial Cells 
The most common mutation in the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) gene, ΔF508, results in the production of a misfolded protein that is rapidly degraded. The mutant protein is temperature sensitive, and prior studies indicate that the low-temperature–rescued channel is poorly responsive to physiological stimuli, and is rapidly degraded from the cell surface at 37°C. In the present studies, we tested the effect of a recently characterized pharmacological corrector, 2-(5-chloro-2-methoxy-phenylamino)-4′-methyl-[4,5′bithiazolyl-2′-yl]-phenyl-methanone (corr-4a), on cell surface stability and function of the low-temperature–rescued ΔF508 CFTR. We demonstrate that corr-4a significantly enhanced the protein stability of rescued ΔF508 CFTR for up to 12 hours at 37°C (P < 0.05). Using firefly luciferase–based reporters to investigate the mechanisms by which low temperature and corr-4a enhance rescue, we found that low-temperature treatment inhibited proteasomal function, whereas corr-4a treatment inhibited the E1-E3 ubiquitination pathway. Ussing chamber studies indicated that corr-4a increased the cAMP-mediated ΔF508 CFTR response by 61% at 6 hours (P < 0.05), but not at later time points. However, addition of the CFTR channel activator, 4-methyl-2-(5-phenyl-1H-pyrazol-3-yl)-phenol, significantly augmented cAMP-stimulated currents, revealing that the biochemically detectable cell surface ΔF508 CFTR could be stimulated under the right conditions. Our studies demonstrate that stabilizing rescued ΔF508 CFTR was not sufficient to obtain maximal ΔF508 CFTR function in airway epithelial cells. These results strongly support the idea that maximal correction of ΔF508 CFTR requires a chemical corrector that: (1) promotes folding and exit from the endoplasmic reticulum; (2) enhances surface stability; and (3) improves channel activity.
PMCID: PMC2830406  PMID: 19502384
cell surface trafficking; cystic fibrosis transmembrane conductance regulator; ΔF508 rescue; short-circuit current
2.  Activation of the Unfolded Protein Response by ΔF508 CFTR 
Environmental insults and misfolded proteins cause endoplasmic reticulum (ER) stress and activate the unfolded protein response (UPR). The UPR decreases endogenous cystic fibrosis transmembrane conductance regulator (CFTR) mRNA levels and protein maturation efficiency. Herein, we investigated the effects of the folding-deficient ΔF508 CFTR on ER stress induction and UPR activation. For these studies, we developed and characterized stable clones of Calu3ΔF cells that express different levels of endogenous wild-type (WT) and recombinant ΔF508 CFTR. We also present a novel RT-PCR-based assay for differential quantification of wild-type CFTR mRNA in the presence of ΔF508 CFTR message. The assay is based on a TaqMan minor groove binding (MGB) probe that recognizes a specific TTT sequence (encoding phenylalanine at position 508 in human CFTR). The MGB probe is extremely specific and sensitive to changes in WT CFTR message levels. In RNA samples that contain both WT and ΔF508 CFTR mRNAs, measurement of WT CFTR mRNA levels (using the MGB probe) and total CFTR mRNA (using commercial primers) allowed us to calculate ΔF508 CFTR mRNA levels. The results indicate that overexpression of ΔF508 CFTR causes ER stress and activates the UPR. UPR activation precedes a marked decrease in endogenous WT CFTR mRNA expression. Furthermore, polarized airway epithelial cell lines are important tools in cystic fibrosis research, and herein we provide an airway epithelial model to study the biogenesis and function of WT and ΔF508 CFTR expressed within the same cell.
PMCID: PMC2551705  PMID: 18458236
endoplasmic reticulum stress; unfolded protein response; ΔF508 CFTR; quantitative PCR; Calu-3
3.  DETANO and Nitrated Lipids Increase Chloride Secretion across Lung Airway Cells 
We investigated the cellular mechanisms by which nitric oxide (NO) increases chloride (Cl−) secretion across lung epithelial cells in vitro and in vivo. Addition of (Z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl) amino] diazen-1-ium-1, 2-diolate (DETANONOate [DETANO];1–1,000 μM) into apical compartments of Ussing chambers containing Calu-3 cells increased short-circuit currents (Isc) from 5.2 ± 0.8 to 15.0 ± 2.1 μA/cm2 (X ± 1 SE; n = 7; P < 0.001). NO generated from two nitrated lipids (nitrolinoleic and nitrooleic acids; 1–10 μM) also increased Isc by about 100%. Similar effects were noted across basolaterally, but not apically, permeabilized Calu-3 cells. None of these NO donors increased Isc in Calu-3 cells pretreated with 10 μM 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (an inhibitor of soluble guanylyl cyclase). Scavenging of NO either prevented or reversed the increase of Isc. These data indicate that NO stimulation of soluble guanylyl cyclase was sufficient and necessary for the increase of Isc via stimulation of the apical cystic fibrosis transmembrane regulator (CFTR). Both Calu-3 and alveolar type II (ATII) cells contained CFTR, as demonstrated by in vitro phosphorylation of immunoprecipitated CFTR by protein kinase (PK) A. PKGII (but not PKGI) phosphorylated CFTR immuniprecipitated from Calu-3 cells. Corresponding values in ATII cells were below the threshold of detection. Furthermore, DETANO, 8-Br-cGMP, or 8-(4-chlorophenylthio)-cGMP (up to 2 mM each) did not increase Cl− secretion across amiloride-treated ATII cells in vitro. Measurements of nasal potential differences in anesthetized mice showed that perfusion of the nares with DETANO activated glybenclamide-sensitive Cl− secretion. These findings suggest that small concentrations of NO donors may prove beneficial in stimulating Cl− secretion across airway cells without promoting alveolar edema.
PMCID: PMC2542453  PMID: 18314534
Calu-3 cells; cystic fibrosis transmembrane conductance regulator; nasal potential difference; protein kinase G type II; alveolar type II cells
4.  VCP/p97 AAA-ATPase Does Not Interact with the Endogenous Wild-Type Cystic Fibrosis Transmembrane Conductance Regulator 
The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel that is defective in cystic fibrosis. The most common mutation, ΔF508 CFTR, is retained in the endoplasmic reticulum, retrotranslocated into the cytosol, and degraded by the proteasome. In a proteomics screen to identify ΔF508 CFTR interacting proteins, we found that valosin-containing protein (VCP)/p97, a Type II AAA ATPase that is a component of the retrotranslocation machinery, binds ΔF508 CFTR, and this interaction is stabilized by proteasomal inhibition. Since wild-type (WT) CFTR has been reported to be inefficiently processed during biogenesis with as much as 75% of the newly synthesized protein degraded by the proteasome, we examined the VCP interaction in Calu-3, T-84, and 16HBE, three epithelial cell lines that endogenously express WT CFTR. The results indicate that when WT CFTR processing is efficient, as demonstrated in Calu-3 cells, VCP does not interact. Interestingly, overexpression of recombinant WT CFTR in Calu-3 cells results in inefficient processing and VCP interaction, demonstrating that CFTR processing efficiency and the VCP interaction are tightly coupled. Furthermore, induction of ER stress and activation of the unfolded protein response result in inefficient processing of WT CFTR in Calu-3 cells and promote the WT CFTR–VCP interaction. The results support the hypothesis that components of the retrotranslocation machinery such as VCP do not interact with CFTR in epithelial cells that endogenously express WT CFTR, since under normal conditions the processing of the WT protein is efficient.
PMCID: PMC1899338  PMID: 17272822
CFTR; p97/VCP; epithelium; ubiquitination; biogenesis

Results 1-5 (5)