Regulatory role of β-arrestin-2 in cholesterol processing in cystic fibrosis epithelial cells

Mary E. Manson; Deborah A. Corey; Ilya Bederman; James D. Burgess; Thomas J. Kelley

doi:10.1194/jlr.M021972

Regulatory role of β-arrestin-2 in cholesterol processing in cystic fibrosis epithelial cells

^*Departments of Chemistry, Case Western Reserve University, Cleveland, OH
^†Pediatrics, Case Western Reserve University, Cleveland, OH

Abstract

Cystic fibrosis (CF) cells exhibit an increase in the protein expression of β-arrestin-2 (βarr2) coincident with perinuclear accumulation of free cholesterol. Arrestins are proteins that both serve as broad signaling regulators and contribute to G-protein coupled receptor internalization after agonist stimulation. The hypothesis of this study is that βarr2 is an important component in the mechanisms leading to cholesterol accumulation characteristic of CF cells. To test this hypothesis, epithelial cells stably expressing GFP-tagged βarr2 (βarr2-GFP) and respective GFP-expressing control cells (cont-GFP) were analyzed by filipin staining. The βarr2-GFP cells show a late endosomal/lysosomal cholesterol accumulation that is identical to that seen in CF cells. This βarr2-mediated accumulation is sensitive to Rp-cAMPS treatment, and depleting βarr2 expression in CF-model cells by shRNA alleviates cholesterol accumulation compared with controls. Cftr/βarr2 double knockout mice also exhibit wild-type (WT) levels of cholesterol synthesis, and WT profiles of signaling protein expression have previously been shown to be altered in CF due to cholesterol-related pathways. These data indicate a significant regulatory role for βarr2 in the development of CF-like cholesterol accumulation and give further insight into cholesterol processing mechanisms. An impact of βarr2 expression on Niemann-Pick type C-1 (NPC1)-containing organelle movement is proposed as the mechanism of βarr2-mediated alterations on cholesterol processing. It is concluded that βarr2 expression contributes to altered cholesterol trafficking observed in CF cells.

Footnotes

↵1 M. E. Manson and D. A. Corey contributed equally to this work.
↵2 To whom correspondence should be addressed. e-mail: thomas.kelley{at}case.edu
Abbreviations:

β2-AR

β2-adrenergic receptor

βarr2

β-arrestin-2

βarr2-GFP

GFP-tagged βarr2 cell

CF

cystic fibrosis

CFTR

cystic fibrosis transmembrane conductance regulator

DKO

double knockout

cont-GFP

GFP-expressing control cell

GPCR

G-protein coupled receptor

GRK

G-protein coupled receptor kinase

MNE

mouse nasal epithelia

NOS2

nitric oxide synthase 2

NPC1

Niemann-Pick type C-1

WT

wild-type
This work was supported by a grant from the Cystic Fibrosis Foundation and by National Institutes of Health Grants HL-080319 and EB-009481. Technical support was provided by core facilities of the Cystic Fibrosis Center (P30-DK-27651). Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the National Institutes of Health.