Submitted on July 9, 2007
Accepted on December 7, 2007
ClC-3 is required for LPA-activated Cl- current activity and Fibroblast to Myofibroblast Differentiation
ZHAOHONG YIN1, yiai tong2, haiqing zhu3, and Mitchell A Watsky4*
1 Physiology, University of Tennessee Health Science Center, Memphis, Tennessee, United States
2 Anatomy and Neurobiology, University of Tennessee Health Science Center, Memphis, Tennessee, United States
3 Pathology, St.Jude Children Reserch Hospital, Memphis, Tennessee, United States
4 Physiology, Univ. of Tennessee Health Science Center, Memphis, Tennessee, United States
* To whom correspondence should be addressed. E-mail: mwatsky{at}physio1.utmem.edu.
To determine the effects of ClC-3 knockdown and overexpression on LPA- and volume-regulated Cl- currents (IClLPA and IClVRAC, respectively), cell differentiation and cell volume regulation, a short hairpin RNA (shRNA) expression system based on a mouse U6 promoter was utilized to knockdown 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 were used to detect ClC-3 mRNA and protein levels. Whole-cell perforated patch-clamp recording was used to measure IClLPA and IClVRAC currents, and FACS analysis was used to measure cell volume regulation. ClC-3 knockdown significantly decreased IClLPA and IClVRAC activity in the presence of TGF-
1 compared to controls, whereas ClC-3 overexpression resulted in increased IClLPA activity in the absence of TGF-1. ClC-3 knockdown also resulted in a reduction of 
-SMA protein levels in the presence of TGF-1, whereas ClC-3 overexpression increased -SMA protein expression in the absence of TGF-1. In addition, keratocytes transfected with ClC-3 shRNA had a significantly blunted RVD response following hyposmotic stimulation compared to controls. These data confirm that ClC-3 is important in VRAC function and cell volume regulation, is associated with the IClLPA current activity, and participates in the fibroblast to myofibroblast transition.
