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Modulation of F508 Cystic Fibrosis Transmembrane Regulator Trafficking and Function with 4-Phenylbutyrate and Flavonoids

Department of Pediatrics, the Johns Hopkins University School of Medicine, Baltimore, Maryland

Address correspondence to: Pamela L. Zeitlin, Department of Pediatrics, Johns Hopkins University School of Medicine, Park 316, 600 N. Wolfe St., Baltimore, MD 21287. E-mail: pzeitlin{at}jhmi.edu

Over 70% of patients with cystic fibrosis have the F508 mutation. This protein is a partially functional chloride (Cl^–) channel that is prematurely degraded in the endoplasmic reticulum. Specific members of the flavonoid class of compounds have been shown to increase Cl^– conductance of wild-type and F508 cystic fibrosis transmembrane regulator (CFTR). Although flavonoid effects on CFTR processing are unknown, evidence of effects on heat shock proteins, specifically those that have been shown to interact with CFTR, led us to believe that there would be an effect on CFTR processing through modulation of CFTR–chaperone interactions. We sought to determine (i) the effect of apigenin, genistein, kaempferol, and quercetin on CFTR processing in IB3–1 cells (F508/W1282X) and (ii) whether sequential treatment with 4-phenylbutyrate (4-PBA) to increase CFTR processing and flavonoid to directly stimulate CFTR would increase Cl^– conductance. Our results show no significant effect on CFTR processing as measured by immunoblotting with 1 µM or 5 µM of apigenin, genistein, kaempferol, or quercetin. However, despite no effect on CFTR processing as determined by immunoblot, immunofluorescence demonstrated a favorable change in the intracellular distribution of CFTR with 24 h treatments of apigenin, kaempferol, and genistein. Furthermore, we observed an increase in Cl^– conductance as measured by Cl^– efflux in cells that were treated for 24 h with 4-PBA and then assayed with forskolin and 1 µM or 5 µM genistein, and also with cells treated for 24 h with either 4-PBA, 5 µM apigenin, or 1 µM quercetin. Thus, a combination of chronic treatment with 4-PBA or select flavonoids, followed by acute flavonoid exposure, may be beneficial in cystic fibrosis.

Abbreviations: bovine serum albumin, BSA • cystic fibrosis, CF • CF transmembrane regulator, CFTR • chloride, Cl^– • heat shock cognate, Hsc • heat shock protein, Hsp • immunoglobulin, Ig • lactated Ringer's solution, LR • 3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide, MTT • 4-phenylbutyrate, 4-PBA • phosphate-buffered saline, PBS • sodium dodecylsulfate, SDS •

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