Rescue of ΔF508-CFTR (Cystic Fibrosis Transmembrane Conductance Regulator) by Curcumin: Involvement of the Keratin 18 Network

Abstract

The most common mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, ⌬F508, causes retention of ⌬F508-CFTR in the endoplasmic reticulum and leads to the absence of CFTR Cl Ϫ channels in the plasma membrane. ⌬F508-CFTR retains some ClϪ channel activity so increased expression of ⌬F508-CFTR in the plasma membrane can restore Cl Ϫ secretion deficiency. Recently, curcumin was shown to rescue ⌬F508-CFTR localization and function. In our previous work, the keratin 18 (K18) network was implicated in ⌬F508-CFTR trafficking. Here, we hypothesized that curcumin could restore a functional ⌬F508-CFTR to the plasma membrane acting via the K18 network. First, we analyzed the effects of curcumin on the localization of ⌬F508-CFTR in different cell lines (HeLa cells stably transfected with wild-type CFTR or ⌬F508-CFTR, CALU-3 cells, or cystic fibrosis pancreatic epithelial cells CFPAC-1) and found that it was significantly delocalized toward the plasma membrane in ⌬F508-CFTR-expressing cells. We also performed a functional assay for the CFTR chloride channel in CFPAC-1 cells treated or not with curcumin and detected an increase in a cAMP-dependent chloride efflux in treated ⌬F508-CFTR-expressing cells. The K18 network then was analyzed by immunocytochemistry and immunoblot exclusively in curcumin-treated or untreated CFPAC-1 cells because of their endogenic ⌬F508-CFTR expression. After curcumin treatment, we observed a remodeling of the K18 network and a significant increase in K18 Ser52 phosphorylation, a site directly implicated in the reorganization of intermediate filaments.With these results, we propose that K18 as a new therapeutic target and curcumin, and/or its analogs, might be considered as potential therapeutic agents for cystic fibrosis.Cystic fibrosis (CF) is a lethal disease caused by defective function of the cftr gene product, the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) (Riordan et al., 1989). CF is characterized by abnormal chloride transport in many tissues, including lung, pancreas, gastrointestinal tract, liver, sweat glands, and male reproductive ducts. The most common mutation, ⌬F508, results in the production of an immature protein, which is retained in the endoplasmic reticulum (ER) for subsequent proteolytic degradation (Ward et al., 1995), preventing the correct localization of functional protein (Cheng et al., 1990;Ward and Kopito, 1994;Ward et al., 1995). However, it has been shown that, in certain tissues, in vivo, a small amount of ⌬F508-CFTR may reach the plasma membrane (Kalin et al., 1999) and function as a cAMP-activated Cl Ϫ channel but with a decreased chloride channel open probability compared with WT-CFTR (Dalemans et al., 1991;Benharouga et al., 2001).Among the different strategies that increase the delivery of ⌬F508-CFTR to the plasma membrane are low temperature, chemical chaperones, such as glycerol, and molecular chaperones, such as 4-phenylbutyrate (Buphenyl) or benzo[c-]quinolizinium components (MPB-07 and MPB-91) (for review see ...

Emergent Properties of Proteostasis in Managing Cystic Fibrosis

“…In this regard, an extensive effort has been devoted to the development of a CF "interaction" network through data-mining efforts provided by GeneGo (CF MetaMiner [CF]) (Wright et al 2009) that provides a carefully annotated, systems level view of components contributing to CFTR function and correction of disease etiology. Moreover, in addition to studies on the WT and DF508-CFTR interactome (Wang et al 2006), a series of bioinformatic studies have analyzed both the genomic and whole cell proteomic responses of human tissue to disease (Lipecka et al 2006;Ollero et al 2006;Pollard et al 2006;Singh et al 2006;Singh et al 2008). Together, these studies illuminate an extensive network of interactions that will need to be restored to obtain full correction of the disease phenotype (Fig.…”

Bcl-2 Suppresses Sarcoplasmic/Endoplasmic Reticulum Ca2+-ATPase Expression in Cystic Fibrosis Airways

“…These investigators have suggested that blockade of this chaperone interaction by the use of SERCA inhibitors allows misfolded DF508 CFTR to escape the ER, reach the cell surface, and function as a Cl 2 channel (3). These findings precipitated a remarkable number of investigations and resulted in several papers, indicating that SERCA pump inhibitors like curcumin and/or thapsigargin can (4)(5)(6)(7)(8)(9) or cannot (10-13) enhance DF508 CFTR trafficking to the plasma membrane and apical epithelial chloride transport.…”

A Cystic Fibrosis Respiratory Epithelial Cell Chronically Treated by Miglustat Acquires a Non–Cystic Fibrosis–Like Phenotype

“…Other small molecules, such as curcuminoids (12)(13)(14), 4-phenylbutyrate (4-PBA, buphenyl) (15,16), and CFTRcor-325 (17), are also potential candidates to rescue F508del-CFTR function. Most of these small molecules act after short-term incubation (2-24 hours, depending on studies and laboratories) of CF cells, and little if any information is available concerning their long-term effect.…”