ERp29 Regulates ΔF508 and Wild-type Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Trafficking to the Plasma Membrane in Cystic Fibrosis (CF) and Non-CF Epithelial Cells

Laurence Suaud,Susan Guttentag,Catherine Kebler,Katelyn Miller,Wusheng Yan,Lora Alvey,Ronald C Rubenstein,Michael J Hubbard,James L Kreindler,Amal Robay

doi:10.1074/jbc.m111.240267

Abstract

Sodium 4-phenylbutyrate (4PBA) improves the intracellular trafficking of ΔF508-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 ΔF508-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 ΔF508-CFTR trafficking. ERp29 mRNA and protein expression was significantly increased (∼1.5-fold) in 4PBA-treated IB3-1 cells. In Xenopus oocytes, ERp29 overexpression increased the functional expression of both wild-type and ΔF508-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, ΔF508-CFTR co-immunoprecipitated with endogenous ERp29, and overexpression of ERp29 led to increased ΔF508-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 ΔF508-CFTR trafficking in CF epithelial cells.

Highlights

Sodium 4-phenylbutyrate (4PBA) improves ⌬F508-CFTR intracellular trafficking in cystic fibrosis (CF) epithelial cells such as the IB3-1 CF human bronchiolar epithelial cell line as early as 4 – 8 h after exposure and restores CFTR function at the plasma membrane without altering CFTR mRNA expression [16]
The development of novel, mechanism-based therapies for CF is founded on the hypothesis that repair of mutant CFTR dysfunction will result in improved clinical outcomes [46]
We have shown that such alterations in Hsc70 and Hsp70 expression can modulate the intracellular trafficking of the epithelial sodium channel ENaC [49]

Summary

Introduction

Sodium 4-phenylbutyrate (4PBA) improves ⌬F508-CFTR intracellular trafficking in CF epithelial cells such as the IB3-1 CF human bronchiolar epithelial cell line (genotype ⌬F508/ W1282X) as early as 4 – 8 h after exposure and restores CFTR function at the plasma membrane without altering CFTR mRNA expression [16]. These data suggest that increased expression of ERp29 may contribute to improved ⌬F508-CFTR trafficking in response to 4PBA and that ERp29 may act as a chaperone of a medically important plasma membrane protein. For experiments examining the influence of ERp29 overexpression on WT- or ⌬F508-CFTR functional expression in oocytes, data at Ϫ100-mV holding potential are presented as mean Ϯ S.E. with significance determined by one-way ANOVA in comparison with oocytes injected with WT- or ⌬F508-CFTR alone.

Results

Conclusion