ClC-3 is required for LPA-activated Cl- current activity and fibroblast-to-myofibroblast differentiation.
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.
Yin
Zhaohong
Z
Department of Physiology, University of Tennessee Health Science Center, 894 Union Ave., Memphis, TN 38163, USA.
Tong
Yiai
Y
Zhu
Haiqing
H
Watsky
Mitchell A
MA
D000199:Actins / Q000378:metabolism; D002454:Cell Differentiation / Q000235:genetics*; D002460:Cell Line; D048429:Cell Size / Q000187:drug effects; D018118:Chloride Channels / Q000235:genetics*; D002712:Chlorides / Q000378:metabolism*; D015536:Down-Regulation / Q000187:drug effects / Q000235:genetics; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D005355:Fibrosis / Q000378:metabolism / Q000503:physiopathology; D005434:Flow Cytometry; D006801:Humans; D015603:Keratinocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D008246:Lysophospholipids / Q000494:pharmacology; D008564:Membrane Potentials / Q000187:drug effects / Q000235:genetics; D032389:Myocytes, Smooth Muscle / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D034622:RNA Interference; D053773:Transforming Growth Factor beta1 / Q000378:metabolism / Q000494:pharmacology; D014945:Wound Healing / Q000502:physiology
C541116:ACTA2 protein, human; D000199:Actins; D018118:Chloride Channels; D002712:Chlorides; C115376:ClC-3 channel; D008246:Lysophospholipids; D053773:Transforming Growth Factor beta1; C032881:lysophosphatidic acid