Sodium 4-phenylbutyrate (4PBA) improves the intracellular trafficking of ⌬F508-CFTR in cystic fibrosis (CF) epithelial cells. The underlying mechanism is uncertain, but 4PBA modulates the expression of some cytosolic molecular chaperones. To identify other 4PBA-regulated proteins that might regulate ⌬F508-CFTR trafficking, we performed a differential display RT-PCR screen on IB3-1 CF bronchiolar epithelial cells exposed to 4PBA. One transcript up-regulated by 4PBA encoded ERp29, a luminal resident of the endoplasmic reticulum (ER) thought to be a novel molecular chaperone. We tested the hypothesis that ERp29 is a 4PBA-regulated ER chaperone that influences ⌬F508-CFTR trafficking. ERp29 mRNA and protein expression was significantly increased (ϳ1.5-fold) in 4PBA-treated IB3-1 cells. In Xenopus oocytes, ERp29 overexpression increased the functional expression of both wild-type and ⌬F508-CFTR over 3-fold and increased wild-type cystic fibrosis transmembrane conductance regulator (CFTR) plasma membrane expression. In CFBE41o؊ WT-CFTR cells, expression of and short circuit currents mediated by CFTR decreased upon depletion of ERp29 as did maturation of newly synthesized CFTR. In IB3-1 cells, ⌬F508-CFTR co-immunoprecipitated with endogenous ERp29, and overexpression of ERp29 led to increased ⌬F508-CFTR expression at the plasma membrane. These data suggest that ERp29 is a 4PBA-regulated ER chaperone that regulates WT-CFTR biogenesis and can promote ⌬F508-CFTR trafficking in CF epithelial cells.
Sodium 4‐Phenylbutyrate (4PBA) improves the aberrant intracellular trafficking of deltaF508‐CFTR in Cystic Fibrosis (CF) epithelial cells. We hypothesized that 4PBA causes a complex cellular adaptation leading to altered expression of molecular chaperones and improved deltaF508 intracellular trafficking. To identify elements of this cellular response, we performed differential display RT‐PCR on RNA extracted from IB3‐1 bronchiolar epithelial cells (genotype deltaF508/W1282X) treated for 0–24 hours with 1 mM 4PBA. We isolated a cDNA clone for ERp29 (29 kDa ER Lumenal Protein) that had a time‐dependant increase in mRNA expression. ERp29 is a ubiquitous, conserved thioredoxin homolog that is prominently expressed in lung. IB3‐1 cells treated with 4PBA had increased ERp29 mRNA expression by ribonuclease protection assays and protein expression by immunoblot. In coimmunoprecipitation experiments, ERp29 was associated with deltaF508‐CFTR in IB3‐1 cell lysates, but not detectably with wt‐CFTR in lysates of T84 intestinal epithelial cells. In oocytes, modest ERp29 overexpression increased the functional expression of wt and deltaF508‐CFTR. This effect was lost at higher levels of ERp29 overexpression. The increased wt‐CFTR functional expression corresponded to increased expression of CFTR at the oocyte surface. These data are consistent with increased ERp29 expression facilitating ΔF508 trafficking and functional expression in CF epithelial cells in response to 4PBA.Supported by grants from NIDDK.
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