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Yin Z, Tong Y, Zhu H, Watsky MA: ClC-3 is required for LPA-activated Cl- current activity and fibroblast-to-myofibroblast differentiation. Am J Physiol Cell Physiol. 2008 Feb;294(2):C535-42. Epub 2007 Dec 12.
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-beta (1) (TGF-beta (1)) compared with controls, whereas ClC-3 overexpression resulted in increased I (Cl,LPA) activity in the absence of TGF-beta (1). ClC-3 knockdown also resulted in a reduction of alpha-smooth muscle actin (alpha-SMA) protein levels in the presence of TGF-beta (1), whereas ClC-3 overexpression increased alpha-SMA protein expression in the absence of TGF-beta (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. |
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