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Am J Physiol Cell Physiol 294: C535-C542, 2008. First published December 12, 2007; doi:10.1152/ajpcell.00291.2007
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MEMBRANE TRANSPORTERS, ION CHANNELS, AND PUMPS

ClC-3 is required for LPA-activated Cl– current activity and fibroblast-to-myofibroblast differentiation

Zhaohong Yin,1 Yiai Tong,2 Haiqing Zhu,3 and Mitchell A. Watsky1

Departments of 1Physiology and 2Anatomy and Neurobiology, University of Tennessee Health Science Center, Memphis, Tennessee; and 3Department of Pathology, St. Jude Children's Research Hospital, Memphis, Tennessee

Submitted 9 July 2007 ; accepted in final form 7 December 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
To determine the effects of chloride channel 3 (ClC-3) knockdown and overexpression on lysophosphatidic acid (LPA)- and volume-regulated anion channel Cl– currents (ICl,LPA and ICl,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 ICl,LPA and ICl,VRAC currents, and fluorescence-activated cell sorting analysis was used to measure cell volume regulation. ClC-3 knockdown significantly decreased ICl,LPA and ICl,VRAC activity in the presence of transforming growth factor-β1 (TGF-β1) compared with controls, whereas ClC-3 overexpression resulted in increased ICl,LPA activity in the absence of TGF-β1. ClC-3 knockdown also resulted in a reduction of {alpha}-smooth muscle actin ({alpha}-SMA) protein levels in the presence of TGF-β1, whereas ClC-3 overexpression increased {alpha}-SMA protein expression in the absence of TGF-β1. 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 ICl,LPA current activity, and participates in the fibroblast-to-myofibroblast transition.

chloride channel-3; lysophosphatidic acid; cornea; lung; keratocyte

MYOFIBROBLASTS ARE CRITICAL components of the wound healing process and are intimately involved in fibrotic pathology. The fibroblast-to-myofibroblast transition is one of the key events in the formation and contraction of granulation tissue (7), a key step in wound healing. Following wound repair, myofibroblasts disappear through selective apoptosis. Persistent myofibroblast proliferation and/or survival represent a pathological repair process, which can result in fibrosis (e.g., pulmonary fibrosis, kidney fibrosis, and liver cirrhosis) and organ failure (7, 11). Myofibroblasts, which are commonly identified by their expression of {alpha}-smooth muscle actin ({alpha}-SMA) and by features that are intermediate between those of smooth muscle cells and fibroblasts, can be regulated by many factors. Cytokines like transforming growth factor (TGF)-β, particularly TGF-β1, can stimulate myofibroblast differentiation from fibroblasts both in vitro (3) and in vivo (18). Inflammatory cells such as macrophages and lymphocytes can produce TGF-β1, as do other cell types, including fibroblasts, epithelial cells, endothelial cells, and platelets. Myofibroblasts themselves can also secrete TGF-β1, establishing a positive-feedback loop for myofibroblast differentiation and the progression of fibrosis (29, 30).

Our lab previously discovered that corneal keratocytes isolated from wounded rabbit corneas or cultured in the presence of serum contain a volume-regulated Cl– current (ICl,LPA) that is also activated, via a receptor-mediated pathway, by the phospholipid growth factors lysophosphatidic acid (LPA) and sphingosine-1-phosphate (24). We also determined that ICl,LPA activity plays a critical role in the differentiation of human lung fibroblasts to myofibroblasts (27). Chloride channels are ubiquitously expressed in almost all eukaryotic cells. They are pore-forming transmembrane proteins that allow the passive transport of Cl– across biological membranes (15). In the plasma membrane, Cl– channels are essential for the regulation of cell volume. Chloride channel 3 (ClC-3), a well-studied member of the ClC transporter and channels superfamily, has been postulated as the volume-regulated chloride channel. It has been shown that ClC-3 antisense reduces ClC-3 expression as well as the swelling-activated Cl– current (ICl,swell) in rabbit nonpigmented ciliary epithelial cells (22). Furthermore, Wang et al. (23) reported that intracellular dialysis with an antibody against ClC-3 inhibited native volume-activated chloride currents in guinea pig cardiac cells and canine pulmonary arterial smooth muscle cells. On the other hand, ClC-3 knockout was ineffective at suppressing ICl,swell activity. Thus, at the protein and molecular level, the volume-regulated anion channel (VRAC) has yet to be positively identified, with considerable controversy still surrounding the possibility that ClC-3 is the volume-regulated chloride channel protein (14, 17, 19, 26).

The present study was designed to use ClC-3 knockdown and overexpression in human corneal keratocytes and human fetal lung fibroblasts to determine whether ClC-3 is associated with the ICl,LPA current and whether ClC-3 participates in the fibroblast-to-myofibroblast transition and in volume regulation of these cells.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cell culture. A human corneal keratocyte cell line (8) was grown in medium M199 (Mediatech, Herndon, VA), with 10% FBS (HyClone, Logan, UT), 1% insulin, transferrin, selenium (ITS + Premix, BD Biosciences; Bedford, MA), and 40 µg/ml gentamicin (GIBCO, Invitrogen; Grand Island, NY) at 37°C and 5% CO2. A human fetal lung fibroblast cell line (IMR-90, finite primary cell line) was obtained from the National Institute on Aging Cell Culture Repository (Coriell Institute for Medical Research, Camden, NJ). Cells were grown in minimum essential medium Eagle's medium (Sigma, St. Louis, MO) with 2 mM L-glutamine (Sigma), 10% FBS (HyClone), and 30 µg/ml gentamicin (GIBCO, Invitrogen) at 37°C and 5% CO2.

Short hairpin RNA. A short hairpin small interfering RNA (shRNA) expression system based on a mouse U6 promoter (28) was used to knock down ClC-3 in the two human cell lines. The shRNA construct contained a 21-nt target CLC-3-specific sequence and its complementary sequence separated by a loop RNA sequence. The complete ClC-3 mRNA sequence (NCBI.nucleotide: AF172729 [GenBank] ) was scanned with GenScript siRNA Target Finder software to select five candidate shRNA constructs. Designed sequences were chemically synthesized as two complementary DNA oligonucleotides, annealed from 91°C to 22°C and ligated to the mU6 vector. To test the efficiency of the candidate shRNA constructs, we developed an enhanced green fluorescent protein (eGFP)-ClC-3 reporter system by the method of Kumar et al. (13), by which ClC-3 was fused with the eGFP gene and inserted immediately after the cytomegalovirus promoter in the pcDNA3 vector (Invitrogen). Using this system, ClC-3 knockdown resulted in a reduction in expression of the eGFP reporter gene, leading to a decrease in fluorescence of the cells observed by microscope. Using this cotransfection system in HEK cells, we performed an initial screening of a large number of different eGFP-ClC-3 and shRNA ratios to determine the optimal parameters for the shRNA experiments. The calcium phosphate method was used to transfect the HEK cells. Cotransfected HEK cells had a high transfection efficiency, with up to 80% of the cells displaying the GFP signal. We narrowed down the shRNA and eGFP-ClC-3 ratios to 10:1, 20:1, and 40:1. As expected, the intensity of the GFP signal was construct dependent, with nonrelevant controls displaying the strongest GFP signal. The shRNA construct displaying the weakest GFP signal was considered to possess the highest activity for knockdown of its target gene. Of the five ClC-3 shRNA constructs we tested, shRNA 337 (ClC-3 shRNA) generated the lowest GFP signal and therefore was considered the most efficient shRNA construct for knockdown of its target sequence. The shRNA-337 was thus used for all subsequent experiments. A 4-bp substituted version of ClC-3 shRNA 337 (mutant ClC-3 shRNA) was used as the ClC-3 shRNA control. The sequences of the five candidate shRNA and mutant shRNA are shown in Table 1. In addition, in some experiments we also used a full-length ClC-3 construct, which was kindly provided by Dr. Deborah Nelson, University of Chicago. Full-length ClC-3 was in a pcDNA3.1 zeo.


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  		Table 1. CIC-3 shRNA sequences

 
Transfection protocol. All transfections were performed using a square electroporator (CUY-21, BEX). For human cornea keratocytes, square electric pulses were applied at 250 V, pulse length 20 ms, 1 pulse, interval 980 ms, followed by 7 V, pulse length 400 ms, 5 pulses, interval 600 ms. For human fetal lung fibroblasts, square electric pulses were applied at 180 V, pulse length 25 ms, 1 pulse, interval 975 ms, followed by 8 V, pulse length 525 ms, 5 pulses, interval 475 ms. Cells were harvested in PBS mixed with plasmids at a ratio of 2 x 106:10 µg. For patch-clamp experiments, pcDNA3.1/Hygro (+) (Invitrogen) was used as a selection vector. Where indicated, shRNA-transfected cells and nontransfected control cells were exposed to 2 ng/ml TGF-β1 (Sigma) to stimulate conversion to the myofibroblast phenotype. We previously determined that TGF-β1-induced myofibroblasts have significantly greater ICl,LPA activity than their nonstimulated counterparts. Cells overexpressing ClC-3 were not exposed to TGF-β1.

RT-PCR. RT-PCR was used to measure ClC-3 knockdown or overexpression. Primers were designed by Primers3 software: ClC-3 forward (1878) 5'-GGG-AAG-GCA-TTT-ATG-AAG-CA-3' and ClC-3 reverse (2127) 5'-CTG-AGG-GCA-AAT-CCC-ACT-AA-3'. Total RNA was extracted from cultured cells using RNA STAT-60 (Tel-Test). DNA was digested using RNase-free DNase I (Invitrogen, Carlsbad, CA) to eliminate genomic DNA contamination. All samples were dissolved in diethylpyrocarbonate (DEPC) water. The concentration and purity of total RNA were determined by measuring the optical density at 260 and 280 nm. The SuperScript III One-Step RT-PCR System (Invitrogen) was used to perform the RT-PCR. Each RT-PCR reaction contained 12.5 µl 2x reaction mix, 0.5 µg total RNA, 5 µM/2.5 µl each sense and antisense primer, and 1 µl SuperScript III RT/Platinum Taq, which were added to DEPC water for a total volume of 25 µl. The conditions of amplification were as follows: cDNA synthesis at 55°C for 60 min, predenaturation at 94°C for 90 s, followed by 25 cycles at 94°C for 30 s, 55°C for 30 s, and 72°C for 30 s, followed by maintenance at 72°C for 10 min, and storage at 4°C. RT-PCR products were analyzed on ethidium bromide-stained 2% agarose gels.

Western blot analysis. Cells were gently scraped off culture plates using a cell lifter and were suspended in cold PBS plus protease inhibitors (Sigma). This mixture was centrifuged at 1,500 rpm and then divided into two Eppendorf tubes, which were centrifuged for an additional 5 min at 12,000 rpm, 4°C. Cells from one tube were lysed in RIPA (25 mM Tris·HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, and 0.1% SDS) lysis buffer plus protease inhibitors, and the protein concentration was determined by using protein assay dye reagent concentrate (Bio-Rad, Hercules, CA). Loading buffer (2x) was added to the second tube of cells, which was heated to 70°C for 20 min. This tube was used for the Western blot analysis. Equal amounts of protein were loaded on each lane, were separated by SDS-PAGE using 8% gel, and were transferred to nitrocellulose. Immunoblotting was performed with the use of an anti-ClC-3 antibody (Alomone, Jerusalem, Israel), a monoclonal anti-{alpha}-SMA antibody (Sigma), and an horseradish peroxidase-conjugated secondary antibody. Enhanced chemiluminescence was used for detection. Western blots were digitally photographed, and blot density was determined by using National Institutes of Health Image software.

Electrophysiology. Cells were patch clamped using the amphotericin whole cell perforated-patch technique (24). For patch-clamp analysis, cells were cotransfected with pcDNA3.1/Hygro (+) and the specified plasmid (ClC-3 shRNA, mutant ClC-3 shRNA, or full-length ClC-3) at a ratio of 1:20. After transfection, cells were selected by plating them in 400 µg/ml hygromycin for 2 days. Briefly, currents were recorded using a patch-clamp amplifier (model 200A, Axon Instruments; Burlingame, CA) and accompanying software (pClamp 8.2, Axon Instruments). Cells were clamped at a holding voltage of 0 mV and were stepped to increasingly depolarized voltages, from –80 to 100 mV in 15-mV steps. Records were capacity compensated by the amplifier circuitry, sampled at 2 kHz, and filtered at 1 kHz. Current density, equal to the peak currents divided by the cell capacitance, was calculated for all cells. ICl,LPA activation was examined following the addition of 10 µM LPA to the bath. The pipette solution contained (in mM) 145 KOH, 120 methanesulfonic acid, 2.5 NaCl, 2.5 CaCl2, 5 HEPES, and 240 mg/ml amphotericin B (Sigma). Unless otherwise noted, the bathing solution contained (in mM) 145 NaCl, 5 KCl, 2.5 CaCl2, 5 glucose, and 5 HEPES.

Cell volume measurements. Fluorescence-activated cell sorting (FACS) was used to examine cell volume regulation following a hyposmotic challenge. Cells were trypsinized, divided between two tubes, centrifuged at 1,000 rpm, and washed (2x) with Ca2+- and Mg2+-free PBS containing 1 mM EDTA and 1% BSA, pH 8.0. One tube of cells was resuspended in 290 mosM NaCl Ringer solution + 2% FBS and served as the 0-min sample; the second tube was resuspended in 220 mosM NaCl Ringer solution + 2% FBS + 10 mg/ml propidium iodide (10 µl). Samples were analyzed at 2 and 15 min. A flow cytometer (BD LSR II system) was used to measure the light-scattering properties of the cells at different time points. An argon laser (488 nm) was used as the probing beam, with red light emission used to exclude the dead, propidium iodide-stained cells during later analysis. Because forward-angle light scatter (FSC) originates in particles with diameters that are larger than the wavelength of the probing light, the FSC serves as an indirect measure of overall cell size (16). FSC distribution histograms for viable cells were analyzed with the use of Flowjo software.

Image acquisition and manipulation. All images were acquired at room temperature with a digital camera. The ultraviolet gel pictures in Fig. 1, A (top and bottom) and B (bottom), were obtained using a Kodak DC 290 digital camera and Photoshop 5.5 acquisition software. A Nikon D70 digital camera was used to take the ultraviolet gel and film pictures of Fig. 1B (top), Fig. 2, and Fig. 3; the acquisition software was Adobe Photoshop CS2, version 9. All images were processed in Adobe Photoshop CS2, version 9, and adjustments were made to the entire image using the "levels" layer effects to adjust contrast and background density.


Figure 1
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  		Fig. 1. RT-PCR results showing chloride channel 3 (ClC-3) mRNA knockdown in human corneal keratocytes (A) and human fetal lung fibroblasts (B). Cells were transfected by electroporation with either ClC-3 short hairpin RNA (shRNA 337), a control shRNA (mutant ClC-3 shRNA), or full-length ClC-3 and were harvested 30 h after transfection. Nontransfected cells were also used as controls. Both mutant ClC-3 shRNA and nontransfected cells had similar ClC-3 mRNA levels, whereas ClC-3 shRNA cell RNA was knocked down by ~50%. Full-length ClC-3 human corneal keratocytes and human fetal lung fibroblasts had ~56% and ~75% increases in ClC-3 mRNA, respectively. Relative blot densities (RD) for ClC-3 mRNA are shown above each figure.

 

Figure 2
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  		Fig. 2. Human corneal keratocyte Western blot results. Cells were electroporated with shRNA or full-length ClC-3 and were harvested 2 days after transfection. A: nontransfected cells had similar ClC-3 protein levels when compared with mutant ClC-3 shRNA cells, whereas ClC-3 shRNA cells had an ~50% decrease in ClC-3 protein levels. Full-length ClC-3 cells had an ~80% increase in ClC-3 protein levels. B: keratocytes treated with transforming growth factor-β1 (TGF-β1) had elevated {alpha}-smooth muscle actin ({alpha}-SMA) expression compared with untreated control cells, which had minimal {alpha}-SMA expression. C: TGF-β1-treated keratocytes also had an ~70% increase in ClC-3 protein compared with keratocytes not treated with TGF-β1. D: cells transfected with ClC-3 shRNA in the presence of TGF-β1 had an ~50% decrease in {alpha}-SMA protein levels compared with nontransfected and mutant ClC-3 shRNA transfected cells, whereas the nontransfected and mutant shRNA transfected cells had similar {alpha}-SMA protein levels. E: full-length ClC-3-transfected cells, in the absence of TGF-β1, had an ~60% increase in {alpha}-SMA protein levels compared with the nontransfected cells.

 

Figure 3
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  		Fig. 3. Human lung fibroblast Western blot results. Cells were electroporated with shRNA or full-length ClC-3 and were harvested 2 days after transfection. A: nontransfected fibroblasts had similar ClC-3 protein levels when compared with mutant ClC-3 shRNA fibroblasts, whereas ClC-3 shRNA cells had an ~50% decrease in ClC-3 protein levels. Full-length ClC-3 cells had an ~200% increase in ClC-3 protein levels. B: ClC-3 shRNA-transfected fibroblasts had an ~60% decrease in {alpha}-SMA protein levels compared with nontransfected and mutant ClC-3 shRNA fibroblasts, whereas full-length ClC-3-transfected fibroblasts had an ~160% increase in {alpha}-SMA protein levels.

 

   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
RT-PCR. RT-PCR results showed a reduction in ClC-3 mRNA levels in shRNA-treated cells and increased ClC-3 mRNA levels in cells overexpressing ClC-3. Fig. 1A shows that ClC-3 shRNA reduced ClC-3 mRNA in the human corneal keratocytes ~50% compared with the mutant ClC-3 shRNA control cells and nontransfected control cells, whereas full-length ClC-3 overexpression increased ClC-3 message expression by 56%. To confirm these results in fibroblasts from a different tissue, we repeated these experiments in lung fibroblasts. Fig. 1B shows similar results in human lung fibroblasts, with ClC-3 shRNA knocking down the ClC-3 mRNA level by ~60%, and full-length ClC-3 overexpression increasing ClC-3 mRNA levels by ~75% when compared with mutant ClC-3 shRNA control cells and nontransfected control cells.

Western blot analysis. Western blotting was performed as a semiquantitative assay to measure ClC-3 and {alpha}-SMA expression at the protein level. Fig. 2A shows that ClC-3 shRNA knocked down human corneal keratocyte ClC-3 protein expression by ~50% compared with the mutant ClC-3 shRNA control and nontransfected control, whereas full length ClC-3 overexpression increased ClC-3 protein expression by ~80% compared with the control cells.

The keratocyte-to-myofibroblast transition is characterized by an increase in {alpha}-SMA expression in myofibroblasts. Fig. 2B shows that keratocytes treated with 2 ng/ml TGF-β1 have elevated {alpha}-SMA expression compared with untreated control cells, which had minimal {alpha}-SMA expression. This demonstrates TGF-β1-induced myofibroblast differentiation. Fig. 2C shows that TGF-β1 treatment of control keratocytes (no shRNA or full-length ClC-3) for 2 days also increased ClC-3 protein expression compared with the untreated cells. Fig. 2D shows that cells transfected with ClC-3 shRNA in the presence of TGF-β1 had a 50% reduction in {alpha}-SMA protein expression compared with TGF-β1-treated nontransfected cells and TGF-β1-treated mutant ClC-3 shRNA control cells, whereas Fig. 2E shows that cells overexpressing ClC-3, in the absence of TGF-β1, have close to 60% more {alpha}-SMA when compared with TGF-β1-treated nontransfected control cells. In a similar fashion, human fetal lung fibroblast cells treated with ClC-3 shRNA had ClC-3 protein expression knocked down by ~50% compared with mutant ClC-3 shRNA control cells; whereas full-length ClC-3 overexpression increased ClC-3 protein expression by ~200% compared with the control cells (Fig. 3A). Fig. 3B shows that ClC-3 shRNA-treated lung fibroblasts had a 60% reduction of {alpha}-SMA protein expression compared with the mutant ClC-3 shRNA control cells and that full-length ClC-3 overexpression increased {alpha}-SMA protein by 160% compared with control. These Western blot data confirm that ClC-3 knockdown can inhibit keratocyte/fibroblast differentiation to the myofibroblast phenotype, whereas ClC-3 overexpression promotes myofibroblast differentiation.

Cl– currents induced by LPA and hypotonic solution. Whole cell perforated patch-clamp recordings of human corneal keratocytes showed minimal ICl,LPA activity in control cells grown in the absence of TGF-β1, although hypotonic NaCl Ringer solution (220 mosM) stimulated a large volume-activated Cl– current (ICl,VRAC), as shown from a representative cell in Fig. 4A. LPA evoked ICl,LPA currents in control cells grown in the presence of TGF-β1 (2 ng/ml) for 2 days; and as in the TGF-β1-free cells, hypotonic Ringer solution stimulated a large ICl,VRAC (Fig. 4B). Figure 4C shows patch-clamp tracings from a representative keratocyte transfected with ClC-3 shRNA and grown for 2 days in the presence of 2 ng/ml TGF-β1. LPA did not stimulate any ICl,LPA activity, and hypotonic NaCl Ringer solution stimulated only a relatively small ICl,VRAC. In contrast, keratocytes transfected with full-length ClC-3, grown without TGF-β1, had large LPA-stimulated ICl,LPA currents (Fig. 4D). These cells also showed spontaneous ICl,LPA activity in the absence of LPA (data not shown). Table 2 shows a summary of the keratocyte patch-clamp data. ClC-3 shRNA-transfected, TGF-β1-treated cells had significantly lower ICl,LPA and ICl,VRAC current densities than control and mutant ClC-3 shRNA-transfected cells. No data are shown for hyposmotically challenged ClC-3-transfected cells, because they did not tolerate these conditions.


Figure 4
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  		Fig. 4. Cl– currents in representative human corneal keratocytes. A: lysophosphatidic acid (LPA; 10 µM) activated minimal Cl– current (ICl,LPA), whereas hypotonic NaCl Ringer solution (220 mosM) stimulated a large volume-regulated anion channel Cl– current (ICl,VRAC) in keratocytes grown with no TGF-β1. B: LPA stimulated ICl,LPA, and hypotonic NaCl Ringer solution stimulated a large ICl,VRAC in a representative keratocyte transfected with mutant ClC-3 shRNA and grown in the presence of TGF-β1 (2 ng/ml) for 2 days. C: LPA did not stimulate any ICl,LPA, and hypotonic NaCl Ringer solution stimulated only a relatively small ICl,VRAC, in a representative keratocyte transfected with ClC-3 shRNA and grown in the presence of TGF-β1 for 2 days. D: LPA stimulated a large ICl,LPA in a representative keratocyte grown without TGF-β1 and transfected with full-length ClC-3.

 

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  		Table 2. Human cornea keratocyte patch-clamp results

 
Human fetal lung fibroblasts had similar patch-clamp results. The ICl,VRAC current density of lung fibroblasts cultured with TGF-β1 (2 ng/ml for 2 days), as determined by exposure to 220 mosM hypotonic solution, was no different than control cells, with mean values ± SE of 69.56 ± 8.52 pA/pF and 79.94 ± 22.13 pA/pF, respectively (n = 6; P > 0.05). The data in Table 3 show that ClC-3 knockdown decreased ICl,LPA activity, whereas ClC-3 overexpression increased ICl,LPA activity, which confirms that ClC-3 is indeed associated with the ICl,LPA current. In addition, ClC-3 knockdown decreased ICl,LPA activity, whereas overexpression increased it over non-TGF-β1-treated controls.


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  		Table 3. Human fetal lung fibroblast patch-clamp results

 
Volume regulation analysis. ClC-3 and its associated ionic currents, ICl,LPA and ICl,VRAC, have been postulated to play a significant role in cell volume regulation. We used FACS to examine the influence of ClC-3 on the keratocyte regulatory volume decrease (RVD) response. Fig. 5 shows flow cytometer results from control, ClC-3 shRNA, and mutant ClC-3 shRNA-transfected human corneal keratocytes before and after hyposmotic challenge. Figure 5A shows that, after cells were exposed to a 220 mosM hypotonic solution, control cell FSC shifted to the right, returning to the control level after 15 min. This demonstrates an RVD. Figure 5B shows similar results with mutant ClC-3 shRNA-transfected cells, although the RVD response is not quite as robust as in the control cells. Cells transfected with ClC-3 shRNA, on the other hand, showed little or no volume recovery 15 min after hyposmotic challenge (Fig. 5C).


Figure 5
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  		Fig. 5. Human corneal keratocytes volume regulation. Keratocytes were electroporated with mutant ClC-3 shRNA or ClC-3 shRNA and were harvested 2 days after transfection; nontransfected cells were used as an additional control. A: control keratocyte forward-angle light scatter (FSC) shifted to the right after cells were exposed to a 220 mosM hypotonic solution, then shifted back to control level after 15 min. These shifts represent cell swelling and a subsequent regulatory volume decrease. B: the FSC of cells transfected with mutant ClC-3 shRNA shifted to the right after cells were exposed to a 220 mosM hypotonic solution, then returned close to control level after 15 min. C: the FSC of cells transfected with ClC-3 shRNA shifted to the right after cells were exposed to a 220 mosM hypotonic solution, and the FSC failed to return to control levels after 15 min, which indicated a loss of the regulatory volume decrease response.

 
Propidium iodide staining (data not shown) demonstrated that ClC-3 shRNA transfected keratocytes were more susceptible to dying following hyposmotic challenge than the mutant ClC-3 shRNA-transfected and control cells. This was likely due to their inability to properly volume regulate. These data confirm that ClC-3 is required for proper volume regulation in human corneal keratocytes.


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Myofibroblast differentiation is a key step during wound healing and the development of fibrosis. We previously found that corneal keratocytes isolated from wounded rabbit corneas or cultured in the presence of serum contain a VRAC channel (ICl,LPA) that is also activated, via a receptor-mediated pathway, by the phospholipid growth factors LPA and sphingosine-1-phosphate (24). We also found that ICl,LPA plays a critical role in the differentiation of human lung fibroblasts to myofibroblasts (27). The present study used human corneal keratocytes and human fetal lung fibroblasts to determine whether ClC-3 is associated with the ICl,LPA current, whether it participates in the fibroblast-to-myofibroblast transition, and whether it is responsible for the VRAC current and volume regulation in these cells.

Almost all eukaryotic cells express chloride channels, and recent studies on ClC-K1, ClC-2, ClC-3, ClC-5, and ClC-7 knockout mice have assisted in the identification of human inherited diseases caused by mutations of some of these chloride channels: myotonia congenita for ClC-1, Bartter disease for ClC-Kb, Dent's disease for ClC-5, and osteopetrosis for ClC-7 (10, 21). These studies have provided direct evidence of the physiological relevance and clinical importance of these chloride channels, making the identification of the molecular species underlying VRAC of utmost importance. This is particularly true given our findings that ICl,LPA and ClC-3 are required for the fibroblast-to-myofibroblast transition, and the importance of myofibroblasts in fibrotic disorders and disease, such as systemic sclerosis.

ClC-3, a ubiquitously expressed member of the ClC transporters and channels superfamily, has been postulated as encoding the VRAC channel (4, 5). Since ClC-3 was first cloned by Uchida et al. (20) in 1994, controversial data have been reported concerning its functional expression. Investigations into the expression levels of ClC-3 mRNA and protein, and corresponding VRAC activity in various tissues, has led to conflicting results regarding its connection to VRAC (2, 17, 20, 26). Experiments supporting ClC-3 as the protein responsible for the VRAC channel include intracellular dialysis, with an antibody against ClC-3 that abolished large volume-sensitive outwardly rectifying whole cell Cl– currents in transfected gpClC-3-GFP NIH/3T3 cells. This anti-ClC-3 antibody also inhibited native volume-activated chloride currents in guinea pig cardiac cells and canine pulmonary arterial smooth muscle cells (5, 6). Furthermore, ClC-3 antisense oligonucleotides have been shown to decrease an endogenous swelling-activated chloride current and the rate of RVD in bovine nonpigmented ciliary epithelial cells (25) and in HeLa cells and Xenopus laevis oocytes (9). In contrast, disruption of the Clcn3 gene in mice did not affect swelling-activated chloride currents as tested in hepatocytes, pancreatic acinar cells (19), or salivary acinar cells (1).

In the current study, ClC-3 shRNA was used to knock down ClC-3 expression and examine its role in ICl,LPA and VRAC activity, in fibroblast-to-myofibroblast differentiation, and in RVD. This shRNA knockdown strategy was chosen because, unlike knockout experiments, its relatively rapid effect on ClC-3 protein levels provides little time for substitute proteins to function in its absence. A ClC-3 overexpression strategy was also used to determine whether we could achieve results opposite to those of the knockdown experiments. Interestingly, although the data were not quantified, we found that the cells overexpressing ClC-3 were more fragile and had a much shorter lifespan than any other cell type we examined. Human fibroblasts from two distinct organ systems were used as a control to confirm that the results were not linked to a specific cell type or line, and also to obtain a better understanding of the functionality of human ClC-3.

RT-PCR results confirmed that ClC-3 shRNA could efficiently reduce ClC-3 mRNA expression by ~50% in both human corneal keratocytes and human fetal lung fibroblasts. Furthermore, Western blot results confirmed a reduction in ClC-3 protein expression in both cell types. We did not expect 100% ClC-3 knockdown because our transfection efficiency was <80%, and because of cell proliferation. To optimize our patch-clamp results, we cotransfected cells with a hygromycin selection vector (pcDNA3.1/Hygro+) at a ratio of 1:20 (hygromycin vector:transfected vector) to ensure that each cell received the transfection vector. Whole cell perforated patch-clamp recordings demonstrated that ClC-3 shRNA cell knockdown of ClC-3 significantly decreased ICl,LPA activity, whereas full-length ClC-3 overexpression significantly increased ICl,LPA activity in the absence of TGF-β1. The absence of TGF-β1 is critical in these overexpression experiments because we previously determined that human lung fibroblasts not exposed to TGF-β1 have minimal ICl,LPA activity (27). Taken together, these results allow us to conclude that ClC-3 is indeed associated with the ICl,LPA current.

The keratocyte-to-myofibroblast transition is characterized by an increase in {alpha}-SMA expression in myofibroblasts. We previously determined that ICl,LPA activity is required for the fibroblast-to-myofibroblast transition, as shown by an inhibition of {alpha}-SMA expression in TGF-β1-treated fibroblasts cultured in the presence of ICl,LPA blockers (27). In the current study, our Western blot results show that human corneal keratocytes grown in the presence of TGF-β1 for 2 days had increased {alpha}-SMA, ClC-3 protein expression, and ICl,LPA activity compared with cells grown with no TGF-β1. Full-length ClC-3 transfected keratocytes and lung fibroblasts had increased ClC-3 and {alpha}-SMA protein level with no TGF-β1 exposure. These results demonstrate that both TGF-β1-treated cells and cells overexpressing ClC-3 have elevated expression of ClC-3 and {alpha}-SMA and increased ICl,LPA activity, whereas ClC-3 knockdown cells have reduced ClC-3 and {alpha}-SMA expression and lower ICl,LPA activity. Because {alpha}-SMA is a myofibroblast-specific protein in these cells, and in view of our previous work indicating the requirement of ICl,LPA activity for the differentiation of fibroblasts to myofibroblasts (27), these results lead us to conclude that ClC-3 participates in keratocyte and fibroblast differentiation to the myofibroblast phenotype.

Our previous studies indicated that ICl,LPA and VRAC are either one or the same, or are at least both activated by cell swelling, and have similar whole cell kinetic features (27). In the current study, we show that ClC-3 knockdown significantly reduced both ICl,LPA and VRAC activity. We also examined the functional role of ClC-3 knockdown on volume regulation in corneal keratocytes. Results from these experiments demonstrate that knocking down ClC-3 results in a significant blunting of the RVD response following hyposmotic stimulation. These results confirm that ClC-3 is required for a normal RVD response in these cells.

In conclusion, the results of ClC-3 shRNA knockdown and full-length ClC-3 overexpression support the hypothesis that ClC-3 is associated with the ICl,LPA current activity, participates in fibroblast-to-myofibroblast differentiation, and is important in VRAC function and cell volume regulation. The identification of ClC-3 as the protein responsible for ICl,LPA and its associated cell functions could be an important step in pharmacologically influencing wound healing and fibrosis in multiple organ systems.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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REFERENCES
 
This work was supported by grants from the National Scleroderma Foundation, the University of Tennessee Rheumatic Disease Core Center (to M. A. Watsky), and Fight for Sight (to Z. Yin).


   ACKNOWLEDGMENTS
 
We thank Victorina Pintea for technical assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. A. Watsky, Dept. of Physiology, Univ. of Tennessee Health Science Center, 894 Union Ave., Memphis, TN 38163 (e-mail: mwatsky{at}physio1.utmem.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
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