In:
American Journal of Physiology-Cell Physiology, American Physiological Society, Vol. 294, No. 2 ( 2008-02), p. C535-C542
Abstract:
To determine the effects of chloride channel 3 (ClC-3) knockdown and overexpression on lysophosphatidic acid (LPA)- and volume-regulated anion channel Cl − currents ( I Cl,LPA and I Cl,VRAC , respectively), cell differentiation, and cell volume regulation, a short hairpin RNA (shRNA) expression system based on a mouse U6 promoter was used to knock down ClC-3 in human corneal keratocytes and human fetal lung fibroblasts. ClC-3 overexpression was achieved by electroporating full-length ClC-3, within a pcDNA3.1 vector, into these two cell lines. RT-PCR and Western blot analysis were used to detect ClC-3 mRNA and protein levels. Whole cell perforated patch-clamp recording was used to measure I Cl,LPA and I Cl,VRAC currents, and fluorescence-activated cell sorting analysis was used to measure cell volume regulation. ClC-3 knockdown significantly decreased I Cl,LPA and I Cl,VRAC activity in the presence of transforming growth factor-β 1 (TGF-β 1 ) compared with controls, whereas ClC-3 overexpression resulted in increased I Cl,LPA activity in the absence of TGF-β 1 . ClC-3 knockdown also resulted in a reduction of α-smooth muscle actin (α-SMA) protein levels in the presence of TGF-β 1 , whereas ClC-3 overexpression increased α-SMA protein expression in the absence of TGF-β 1 . In addition, keratocytes transfected with ClC-3 shRNA had a significantly blunted regulatory volume decrease response following hyposmotic stimulation compared with controls. These data confirm that ClC-3 is important in VRAC function and cell volume regulation, is associated with the I Cl,LPA current activity, and participates in the fibroblast-to-myofibroblast transition.
Type of Medium:
Online Resource
ISSN:
0363-6143
,
1522-1563
DOI:
10.1152/ajpcell.00291.2007
Language:
English
Publisher:
American Physiological Society
Publication Date:
2008
detail.hit.zdb_id:
1477334-X
SSG:
12
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