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PKC Isoform–Specific Enhancement of Capacitative Calcium Entry in Human Corneal Epithelial Cells

¹From the Department of Biological Sciences, College of Optometry, State University of New York, New York, New York; the ²Department of Ophthalmology, Columbia University, New York, New York; and the ³Eye Clinic, Charité University Medicine, Berlin, Germany.

	Abstract

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PURPOSE. To determine in human corneal epithelial cells (HCECs) the role of protein kinase C (PKC) in mediating epidermal growth factor (EGF)–induced stimulation of store-operated channel (SOC) activity and capacitative calcium entry (CCE).

METHODS. Single-cell Ca²⁺ fluorescence imaging of fura2-loaded HCECs was used to evaluate CCE. PKC translocation induced by EGF or PDBu was monitored by Western blot analyses of four different subcellular fractions. Plasma membrane Ca²⁺ influx was measured by Mn²⁺ quench rates of fura2-fluorescence. The whole-cell patch clamp configuration was used to determine the SOC activation induced by EGF.

RESULTS. EGF-induced increases in SOC currents through PKC stimulation, since calphostin C inhibited this response. To determine which PKC isoforms mediated EGF-induced increases in CCE, the PKC isoform enrichment of a plasma membrane–containing fraction was determined. From 5 to 30 minutes, its rank order of enrichment was: > ßI > . Preferential PKC and PKCß translocation was in accordance with other results showing that rottlerin and hispidin have the highest efficacy in suppressing EGF-induced CCE augmentation. Furthermore, after PKCß and PKC siRNA knockdown of gene and protein expression, declines in EGF-induced increases in CCE matched those obtained after exposure to a corresponding selective PKC isoform inhibitor.

CONCLUSIONS. EGF-induced PKC stimulation in HCECs mediates SOC activation. This response contributes to CCE, which preferentially depends on PKC and PKCß isoform stimulation. This rank order is based on the findings that either selective knockdown of their expression or exposure to PKC and PKCß isoform inhibitors elicited the largest declines in EGF-augmented CCE.

EGF receptor–mediated control of cell signaling dynamics and kinetics are dependent on interactions among parallel signaling limbs activated by this growth factor. These interactions are referred to as crosstalk, and, through the modulation of signaling strength, they can affect a response linked to receptor activation. One such signaling pathway is the extracellular regulated kinase (ERK) limb of the mitogen-activated protein kinase (MAPK) cascade. Its signaling strength and duration affect the magnitude of EGF-induced stimulation of proliferation. One of the factors that determine ERK limb control of this response is its interaction with a parallel signaling pathway stimulated by EGF-induced increases in cyclic adenosine monophosphate (cAMP). Such an effect activates protein kinase A (PKA), which in turn dampens growth factor–induced ERK limb stimulation of cell cycle progression and proliferation.³ Therefore, studies on EGF-induced cell signaling can, in the clinical setting, lead to the development of novel strategies that optimize cell proliferation and migration so as to hasten injury-induced corneal epithelial wound healing.

EGF receptor–linked cell signaling in corneal epithelial cells entails the activation of myriad cell-signaling pathways.^{4 5 6 7 8 9 10 11} Those directly involved in activating calcium signaling entail the stimulation of phospholipase C (PLC), which generates two second messengers, inositol 1,4,5-trisphosphate (InsP₃) and diacylglycerol (DAG), from membrane phospholipid. IP₃ diffuses rapidly within the cytosol and binds to InsP₃ receptors on the endoplasmic reticulum (ER), which function as calcium channels to release and thereby deplete calcium from lumenal ER stores. The increases in DAG levels combined with transient increases in intracellular [Ca²⁺]_i selectively stimulate DAG and Ca²⁺-dependent PKC isoform activity. In addition, emptying of such stores in turn induces a feedback message of unknown origin to the plasma membrane resulting in the opening of store-operated channels (SOCs).^{12 13} Their activation causes an increase in external calcium entry into the cytosol through a process called capacitative calcium entry (CCE). There is no information in corneal epithelial cells regarding the role of specific PKC isoforms as mediators of EGF-induced CCE augmentation.

In human corneal epithelial cells (HCECs), we have identified gene expression of five different transient receptor potential (TRP) isoforms within the canonical subfamily. Isoforms in this subfamily are designated TRPC and include TRPC1, TRPC3, TRPC4, TRPC6, and TRPC7.¹⁴ At the least, protein expression of the TRPC isoform designated TRPC4 is essential for EGF-induced SOC activation, CCE, and mitogenesis because knockdown of its expression either suppressed or fully blocked each of these responses to EGF. It is conceivable that kinase-mediated protein phosphorylation could also play a role in regulating SOC and CCE because EGF also activates PKC in corneal epithelial cells.^{4 15} Some suggestive evidence for a kinase-induced change in phosphorylation status mediating regulation comes from a recent report in which it was shown in COS7 cells that EGF receptor stimulation activates SOC through tyrosine kinase interaction with TRPC4.¹⁶ An involvement for PKC could be complex because six different PKC isoforms— PKC, PKCßI, PKCßII, PKC, PKC, and PKCµ—were identified in HCECs.¹⁷ At this point, nothing is known regarding the possible roles of these particular PKC isoforms in regulating SOC activation and CCE in these cells.

Protein kinase C is composed of a family of serine-threonine kinases that modulate the function of a variety of signal transduction pathways controlling cell growth, cell differentiation, and wound healing. The PKC gene family is divided into three subgroups based on sequence homology and cofactor requirements: classic-conventional PKC isozymes (, ßI, ßII, and ), which are Ca²⁺-dependent and diacylglycerol (DAG)–stimulated kinases; novel Ca²⁺-independent PKCs (, , , and ), which are Ca²⁺-independent and DAG-stimulated kinases; and atypical Ca²⁺- and lipid-independent PKCs (, , µ, and ), which are Ca²⁺- and DAG-independent kinases.¹⁸ All isoforms express distinct enzymologic properties, differential translocation to intracellular loci upon stimulation through distinct modes of cellular regulation, and unique substrate specificity.

We report here in HCECs that EGF induces SOC activation through direct stimulation of PKC activity. Such increases appear to be mediated by unique PKC isoforms based on the ability of relatively selective PKC inhibitors to decrease EGF stimulation of CCE. The declines in CCE after siRNA knockdown of the candidate isoforms are consistent with those obtained with isoform-selective drug inhibitors. PKC plays a major role in eliciting CCE increases in response to direct stimulation of PKC, whereas EGF-induced stimulation of this response is primarily mediated by both PKC and PKCß isoforms.

	Materials and Methods

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Cell Culture
SV40-immortalized HCECs (a generous gift from Kaoru Araki-Sasaki) were cultured in Dulbecco modified Eagle medium/F12 (Invitrogen, Carlsbad, CA), which contained 6% FBS, 5 ng/mL EGF, 5 µg/mL insulin, and 40 µg/mL gentamicin.¹⁹ Before experimentation, the cells were kept subconfluent and grown for 1 to 2 days in an atmosphere containing 5% CO₂, 95% ambient air at 37°C. To optimize cell responsiveness to EGF, cells were serum starved for 24 hours before experimentation.

[Ca²⁺]_i Imaging
Single-cell fluorescence imaging was performed after fura2-AM (2 µM) loading on the stage of an inverted microscope. Cells were subcultured on 22-mm–diameter circular coverslips (Fisher Scientific, Pittsburgh, PA) and dye loaded at 37°C for 30 minutes with or without PKC inhibitors. The coverslip formed the base of a chamber that was placed on the stage of an inverted microscope (Diaphot 200; Nikon, Tokyo, Japan). The coverslips were washed three times with NaCl Ringer containing (in mM): 141 NaCl, 4.2 KCl, 0.8 CaCl₂, 2 KH₂PO₄, 1 MgCl₂, 5.5 glucose, and 10 HEPES (osmolarity 300, pH 7.4). Before inducing CCE, the cells were first preincubated for 10 minutes with a Ca²⁺-free counterpart supplemented with 0.5 mM EGTA. The cells were alternately illuminated at 340 and 380 nm, and their emission was monitored every 5 seconds at 510 nm with a charge-coupled device (CCD) camera (Roper Scientific, Tucson, AZ). The field of interest contained 24 to 45 cells, and a mean running ratio was calculated for each region. The changes in [Ca²⁺]_i were determined with image-analysis software (Ratiotool; Isee Imaging, Durham, NC).

CCE was first activated by depleting intracellular calcium in Ca²⁺-free medium and inhibiting ER calcium pump activity with 5 µM cyclopiazonic acid (CPA). Plasma membrane calcium influx through pathways that included SOC was evaluated based on increases in [Ca²⁺]_i resulting from 1 mM calcium addback to the bathing medium. Relatively selective PKC isoform inhibitors were used to suppress CCE stimulation in the presence or absence of either 1 µM PDBu or 20 ng/mL EGF.

Mn²⁺ Quenching
CCE was assessed based on measurements of Mn²⁺-induced fluorescence quenching of fura-2 using Dulbecco phosphate buffer containing 500 µM Mn²⁺ Fluorescence quenching resulting from excitation at 360 nm was measured at an emission wavelength of 510 nm. Mn²⁺ influx was quantified by measuring in a spectrofluorometer the normalized rate (slope) of fura-2 quenching. Under this condition, there is a linear relationship between quenching and [Mn²⁺]_i, which was used to determine the rate of Mn²⁺ accumulation and plasmalemma Mn²⁺ permeability.

Western Blot Analyses
An equal amount of protein from the cell lysates was applied to a 10% gradient SDS-polyacrylamide gel and electrophoresed. To resolve PKC isoform profiles in the different fractions, a fixed percentage of the total volume for each of the four fractions was used for analysis. For example, assume that one tenth of the cytosolic containing fraction contained 10 µg protein; in each case, then, 10% of the total volume was used.¹⁷ Gel-separated proteins were transferred to a polyvinylidene fluoride (PVDF) membrane. Membranes were blocked with 5% nonfat skim milk in phosphate-buffered saline containing 0.1% Tween-20 (PBST) for 1 hour and then incubated with isozyme-specific anti–PKC antibodies to PKC, PKC, and PKC (1:1000 dilution; BD Biosciences, Franklin Lakes, NJ) and PKCßI, PKCßII, and PKCµ (1:1000 dilution; Santa Cruz Biotechnology, Santa Cruz, CA) at 4°C overnight. The membranes were washed three times with PBST and incubated with horseradish peroxidase–conjugated secondary antibodies (1:2000 dilution; Santa Cruz Biotechnology) for 1 hour at room temperature. Bound antibody was evaluated using an enhanced chemiluminescence (ECL) detection system (GE Healthcare, Little Chalfont, Buckinghamshire, UK). An anti-ß-actin monoclonal antibody (Santa Cruz Biotechnology) was used to test for equal protein loading. Resolved bands were quantified with NIH software (ImageJ, version 1.61; available by ftp at zippy.nimh.nih.gov/ or at http://rsb.info.nih.gov/nih-image; developed by Wayne Rasband, National Institutes of Health, Bethesda, MD). Results are representative of three independent experiments.

Subcellular Fractionation
HCECs were lysed and subjected, with modification, to subcellular fractionation followed by Western blot analysis with six PKC isozyme-specific antibodies. Serum-starved HCECs were treated with 20 ng/mL EGF or 1 µM PDBu for 30 seconds to 30 minutes; untreated HCECs were the control. Subsequent steps were performed as described earlier.^{17 20} Cells were washed twice with ice-cold PBS and scraped into a homogenization buffer. Homogenization buffer components were 25 mM Tris HCl (pH 7.4), 2 mM EDTA, 10 mM ß-mercaptoethanol, 10% glycerol, 10 µg/mL aprotinin, 10 µg/mL leupeptin, and 1 mM phenylmethylsulfonyl fluoride (PMSF). Cells were allowed to swell for 10 minutes and then were homogenized with approximately 30 strokes in a tight-fitting pestle (Dounce homogenizer; Bellco Glass Co., Vineland, NJ). The homogenates were centrifuged at 500g for 5 minutes, and the low-speed supernatant was centrifuged at 100,000g for 30 minutes. High-speed supernatant constituted the cytosolic fraction. The high-speed pellet was extracted in ice-cold homogenization buffer containing 1% Triton X-100 for 30 to 60 minutes. The Triton-soluble component (membrane fraction) was separated from the Triton-insoluble material (cytoskeletal fraction) by centrifugation at 100,000g for 15 minutes. The cytoskeletal fraction was resuspended in the same buffer and dispersed by sonication. The low-speed pellet containing nuclei and unbroken cells was resuspended in a nuclear buffer. Nuclear buffer components were 25 mM Tris HCl (pH 7.4), 3 mM MgCl₂, 1 mM PMSF, 10 mM ß-mercaptoethanol, and 0.05% Triton X-100. To remove contaminating membrane components, the low-speed pellet homogenate was centrifuged for 5 minutes at 500g, resuspended in the nuclear buffer without Triton X-100, layered over 45% sucrose, and centrifuged at 1900g for 30 minutes. Purified nuclei were resuspended in the homogenization buffer containing 1% Triton X-100 for 30 to 60 minutes. The small amount of insoluble material was removed by centrifugation at 100,000g for 15 minutes at 4°C, and the supernatant was the nuclear fraction. Protein concentration was measured with a bicinchoninic acid assay (Micro BCA protein assay kit; Pierce Biotechnology, Rockford, IL).

siRNA PKCß or PKC Knockdown
PKCß or PKC siRNA was transfected into HCECs with transfection reagent used according to the manufacturer’s instructions (Santa Cruz Biotechnology). All experimental measurements were performed 72 hours after transfection. Immunoblot analysis was performed to evaluate the extent of knockdown of PKCß or PKC protein expression. Nontargeting siRNA was used as a control for monitoring non–sequence-specific effects.

Whole-Cell Patch Clamp
Coverslips with HCECs were mounted on the stage of an upright microscope (BX50WI; Olympus, Tokyo, Japan) for patch-clamp recordings. Cells were superfused with a sodium- and potassium-free extracellular bath solution containing 120 mM N-methyl-D-glucamine, 5.4 mM CsCl, 1.0 mM MgCl₂, 10 mM glucose, 10 mM HEPES acid, and 0.5 mM EGTA (pH adjusted to 7.2). In addition, the bath solution contained 5 µM nifedipine to block voltage-dependent L-type channel activity. To isolate inward currents through Ca²⁺-permeable cation channels, Ca²⁺ (5 mM) was used as a charge carrier. Pipettes of soft glass with a resistance of 2 to 5 M were pulled with a Universal Puller (Sutter Instruments, Novato, CA). Pipettes for whole-cell recordings were filled with a solution containing 130 mM CsCl, 4.0 mM MgCl₂, 10 mM EGTA, and 10 mM HEPES salt (pH adjusted to 7.2). In addition, the pipette solution contained 0.1 mM DIDS (4,4'-diisothiocyanostilbene-2,2'-disulfonic acid), to inhibit possible chloride channel activity. Membrane currents were recorded with an amplifier (EPC 8; HEKA, Lamprecht, Germany). Electrical stimulation, data storage, and processing were performed with TIDA software (HEKA) in conjunction with a PC/AT-compatible computer. All electrophysiologic experiments were performed at room temperature. Membrane capacitances and access resistances were calculated from the capacitative current transient induced by –120 mV hyperpolarization from the holding potential (0 mV) of 300-ms duration. Mean access resistances of 28 ± 3 M (n = 13) and mean membrane capacitances of 59 ± 4 pF (n = 13) were measured in the whole-cell configuration in HCECs. Pipette and membrane capacitances and access resistances were compensated with a patch-clamp amplifier (EPC 8; HEKA). If drugs were added to the bath solution, the concentration was kept below 1:10,000, which did not affect the patch-clamp recordings (data not shown). Whole-cell cation channel currents were recorded for 300 ms using voltage steps ranging between –120 and +20 mV (20-mV increments). The holding potential (HP) was set to 0 mV to eliminate any possible contributions by voltage-dependent calcium channel activity.

Materials
The following materials were used: Dulbecco modified Eagle medium/F12, fetal bovine serum, and phosphate-buffered saline (Invitrogen-Gibco, Grand Island, NY); fura2-AM (Molecular Probes, Eugene, OR); epidermal growth factor and insulin (Upstate Biotechnology, Lake Placid, NY); siRNA for PKCß and PKC (Santa Cruz Biotechnology); HBDDE and PKC V1–2 (Biomol, Plymouth Meeting, PA); hispidin and rottlerin (Calbiochem, La Jolla, CA); gentamicin, cyclopiazonic acid (CPA), phorbol 12, 13-dibutyrate (PDBu), phorbol 12-myristate 13-acetate (PMA), Calphostin C, 4-phorbol didecanoate (4-PDD), and other reagents (Sigma-Aldrich, St. Louis, MO).

Statistical Analysis
Twenty-four to 45 cells were evaluated per experiment. All experiments were repeated three to six times. The figures show typical responses in each experiment. The n values provided indicate the number of experiments per data point. Values are shown as the mean ± SE. Statistical significance was determined by Student’s unpaired t-test or one-way ANOVA, and P < 0.05 was considered significant.

	Results

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EGF and PKC Augmentation of CCE
We used the Ca²⁺ addback protocol to evaluate the role of SOC activation in inducing CCE.⁵ With this protocol, the cells were initially exposed to Ca²⁺-free NaCl Ringers containing 0.5 mM EGTA, and the intracellular calcium store was depleted through exposure to 5 µM CPA. Subsequently, the cells were reexposed to NaCl Ringers medium containing 1 mM Ca²⁺. The resultant increase, shown in Figure 1 of the F₃₄₀/F₃₈₀ ratio, was reflective of CCE. To assess the individual effects of PKC or EGF receptor stimulation on this response, the cells were exposed to 1 µM PDBu or 20 ng/mL EGF at the time of Ca²⁺ addback. Magnitudes of these responses in part resulted from SOC activation. Direct PKC stimulation with PDBu induced a 3.5-fold increase in the F₃₄₀/F₃₈₀-nm ratio. Such an increase was the largest of the shown responses, whereas the EGF increase was intermediate between that of PDBu and the control response resulting solely from restoration of 1 mM Ca²⁺ to the NaCl Ringers medium. The trace representative of the smallest of the four increases in CCE was obtained from cells that had undergone PKC downregulation. This condition resulted from preincubation of the cells for 24 hours with 1 µM PMA. Furthermore, an increase in PKC activity was a component of the control CCE response, because PKC downregulation diminished it below the baseline.

View larger version (18K): [in this window] [in a new window]   FIGURE 1. EGF and PDBu stimulate CCE. HCECs were initially exposed to Ca2+-free NaCl Ringers containing 0.5 mM EGTA. CPA (5 µM) was applied (arrow). The period of supplementation with Ca2+ (1 mM) is indicated by the horizontal bar. Stimulation of HCECs with 1 µM PDBu caused an increase in the calcium addback response compared with that of the control. This augmentation of CCE was greater than that induced by EGF (20 ng/mL). PKC downregulation was obtained through exposure to 1 µM PMA for 24 hours. After suppression of basal PKC activity, the CCE response was less than that obtained by Ca2+ addback in the presence or absence of EGF and PDBu (n = 6 for each condition).

View larger version (22K): [in this window] [in a new window]   FIGURE 2. Suppression of EGF- or PDBu-induced CCE by PKC isoform inhibitors. After preincubation for 30 minutes with hispidin (2 µM), rottlerin (3 µM), PKC V1–2 (10 µM), or HBDDE (50 µM), cells were exposed continuously to 20 ng/mL EGF (A) or 1 µM PDBu (B). Rank order of suppression of EGF augmentation of CCE was rottlerin = hispidin > HBDDE = PKC V1–2. On the other hand, for PDBu enhancement the ranking was rottlerin > hispidin = HBDDE = PKC V1–2. Therefore, EGF augmentation of CCE was preferentially mediated by PKC and PKCß, whereas with PDBu enhancement PKC was more effective than any of the other three isoforms (n = 5 or 6).

Given that our goal was to assess the role of PKC in mediating EGF stimulation of plasma membrane–originated SOC activity and CCE, we focused on changes in PKC content of the plasma membrane–enriched fraction. Over the period of exposure to EGF, notable increases were observed in the M content of PKC and PKCßI isoforms. In addition, the PKC and PKC content increased less than that of PKC and PKCßI. On the other hand, the M content of PKCßII and PKCµ did not appreciably change. Figure 3 shows that the M PKCßI isoform content increased the most (i.e., greater than twofold) over 30 minutes during exposure to EGF. With PKC, its density in M increased somewhat, with a magnitude and time course similar to those obtained with PKCßI. In the Ck fraction, the time course of the increases in PKCßI isoform mirrored those in the M-enriched fraction. Consistent with the increases in the PKCßI content of the M- and Ck-containing fractions, the time course of the declines in PKCßI nuclear and cytosolic fraction content mirrored those in the Ck- and M-enriched fractions.

View larger version (40K): [in this window] [in a new window]   FIGURE 3. Western blot analysis of EGF-induced PKC isoform translocation. Top: Western blot analysis of time-dependent changes in PKC isoform (i.e., , ßI, ßII, , , and, µ) distribution in four subcellular enriched fractions: plasma membrane (M); cytosol (C); cytoskeleton (Ck); nuclear (N). Bottom: time-dependent changes in PKC isoform distribution profile in each of the four fractions subsequent to initiation of exposure to 20 ng/mL EGF. Data are the mean ± SE of results of three independent experiments.

Figure 4 shows the pattern of changes in PKC localization induced by PDBu in each of the four subcellular fractions. The isoform-specific increases in the M fraction induced by PDBu of PKCßI and PKC were mirrored by corresponding decreases in the C fraction. In the Ck and N fractions, PKC and PKCßI increased during exposure to PDBu. After 30 minutes of exposure, the PDBu-induced PKC and PKCßI increases into the M fraction reached 4- and 2.5-fold, respectively. These results are consistent with those shown in Figure 2 in which the relatively selective PKC inhibitor, rottlerin, had a maximum inhibitory effect on EGF and PDBu-induced stimulation of CCE. Similarly, the PKCßI, II inhibitor hispidin had the second largest inhibitory effect on these responses, which is in accordance with PKCßI translocation to the M fraction and which exceeded that of all other isoforms except PKC. The DR lane in Figure 4 shows the results of downregulation after exposure to PMA for 24 hours.

View larger version (40K): [in this window] [in a new window]   FIGURE 4. Western blot analysis of PDBu-induced PKC isoforms translocation. Top: Western blot analysis of time-dependent, PDBu-induced changes in PKC isoform distribution in four subcellular enriched fractions: plasma membrane (M); cytosol (C); cytoskeleton (Ck); nuclear (N). Bottom: time-dependent changes in PKC isoform distribution profile in each of the four fractions subsequent to initiating exposure to 1 µM PDBu. Exposure of HCECs to PMA for 24 hours significantly downregulated PKC isoform expression. DN indicates downregulation (right lanes). Data are the mean ± SE of results of three independent experiments.

PKCß or PKC Knockdown Suppression of EGF- or PDBu-Induced CCE

View larger version (25K): [in this window] [in a new window]   FIGURE 5. siRNA knockdown of PKCß and PKC protein expression. Lane 1: nontransfected cells (control); lane 2: cells transfected with siCONTROL (siCON; nontargeting siRNA); lane 3: cells transfected with siRNA PKCß or PKC. Immunoblot analyses showed that PKC siRNA effectively decreased PKC protein expression relative to their controls (*P < 0.05). Data are expressed as the mean ± SE (n = 3).

View larger version (21K): [in this window] [in a new window]   FIGURE 6. EGF- or PDBu-induced CCE augmentation after PKCß and PKC siRNA knockdown. Cells were exposed continuously to 20 ng/mL EGF or 1 µM PDBu from the time that Ca2+ supplementation was initiated. EGF augmentation of the CCE response was abolished after siRNA knockdown of either PKCß or PKC protein expression (n = 4). After PKC knockdown, enhancement of the CCE response by PDBu was more suppressed than that in cells transfected with PKCß siRNA. Nevertheless, CCE was more suppressed after downregulation than after exposure to PKC or PKCß siRNA (n = 6). The CCE response was unchanged from that of control in cells transfected with irrelevant siRNA (panels A and B) (n = 4).

EGF- and PDBu-Induced Increases in Mn²⁺ Influx through PKCß or PKC Stimulation

View larger version (21K): [in this window] [in a new window]   FIGURE 7. Mn2+ quench of fura2 fluorescence after exposure to EGF. After depletion of ICS calcium with 5 µM CPA in Ca2+-free medium containing 0.5 mM EGTA, 0.5 mM Mn2+ was added in EGF-containing (A) or PDBu-containing (B) bathing medium (n = 6). Also shown are the curves obtained from cells after PKCß or PKC siRNA transfection (n = 4). Cells transfected with siCONTROL (siCON) do not affect EGF- or PDBu-induced increases at the rate of Mn2+ quench (n = 4). The rank order of suppression of EGF augmentation of CCE was PKCß > PKC knockdown (A). On the other hand, for PDBu enhancement, ranking was reversed (B).

PKC-Induced SOC Activation
EGF induces in HCECs increased SOC activity consisting of TRPC4 proteins.¹⁴ Our objective was to determine whether EGF-induced increases in CCE and SOC are associated with PKC stimulation. To make this assessment, we measured EGF-induced SOC currents in the presence and absence of 1 µM calphostin C using the whole-cell patch clamp technique. Cells were exposed to a solution lacking Na⁺ and K⁺ to eliminate any currents that could result from their presence. The solution also contained 5 µM nifedipine to block voltage-dependent L-type Ca²⁺ channel activity. The pipette solution contained 0.1 mM DIDS to inhibit chloride channel activity. Ca²⁺-permeable cation channel inward currents were detected when Ca²⁺ (5 mM) was used as charge carrier after ICS depletion with 10 µM CPA.

Figures 8A and 8B show the experimental configuration used to characterize EGF-induced stimulation of SOC in the presence and absence of calphostin C. Figures 8C and 8D show the currents normalized by cell capacitance in the absence and presence of EGF (20 ng/mL). Figure 8E shows the I-V relationship and reveals that at –120 mV SOC, the normalized maximum current amplitude was 36 ± 6 pA/pF, and EGF increased by 2.5-fold to 98 ± 15 pA/pF (± SEM; n = 3–4; P = 0.0223). Figure 9C shows the current density voltage relationship wherein the current was normalized to capacitance to obtain current density. In the presence of 1 µM calphostin C and bath calcium, the maximum inward current density was 47 ± 17 pA/pF in the absence of EGF and 51 ± 23 pA/pF pA/pF in its presence (n = 3; P = 0.89; Figs. 9A 9B ). Namely, the 2.5-fold EGF-induced increase in these currents was fully suppressed by calphostin C, whereas this PKC inhibitor had no nonspecific effects on the control currents. The fact that these curves were indistinguishable from one another suggests that EGF-induced SOC stimulation was dependent on increases in PKC activity. Figure 9D summarizes the individual and combined effects of EGF and calphostin C on currents induced by SOC activation at –120 mV. Therefore, EGF-induced activation of SOC was mediated through PKC stimulation by this growth factor.

View larger version (6K): [in this window] [in a new window]   FIGURE 8. EGF-mediated SOC stimulation. (A) Experimental design. (B) Voltage pulse protocol. (C) Depolarization pulse induced currents after establishing the whole-cell configuration (control; n = 3). (D) Currents induced after extracellular application of EGF (20 ng/mL; n = 4). (E) Current-voltage relationship (I-V plot). After breaking into the whole-cell configuration (control; filled circles), no SOC currents were observed (n = 3). After extracellular application of EGF (filled rhombs), a significant increase in cation channel current amplitudes could be observed (n = 4). Ca2+ (5 mM) was used as charge carrier. HP, holding potential set to 0 mV.

View larger version (9K): [in this window] [in a new window]   FIGURE 9. PKC inhibition suppressed EGF-induced SOC activation. (A) Currents after establishing the whole-cell configuration (control) in HCECs preincubated with calphostin C (1 µM; n = 3). (B) Currents after extracellular application of EGF (20 ng/mL; n = 3). (C) Current-voltage relationship of the experiments shown in (A) and (B). After breaking into the whole-cell configuration (control; filled circles), no significant increases in EGF-induced cation channel currents were observed in HCECs preincubated with 1 µM calphostin C (n = 3). Ca2+ (5 mM) was used as charge carrier. HP was set to 0 mV. (D) Summary showing that calphostin C completely inhibited EGF-induced increase in SOC activity.

	Discussion

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We monitored the selective involvement of PKC, PKCß, PKC, and PKC activation in mediating CCE. These isoforms were targeted because they were identified in HCECs, and relatively selective inhibitors have been described for only these.^{17 22 25 26 27} The results shown in Figure 1 indicate that EGF-induced stimulation of CCE depends on PKC activation because after the downregulation of PKC activity, CCE enhancement of this response by this growth factor was obviated. The CCE response after downregulation was less than that in other cells not exposed to EGF, suggesting that the CCE response itself was in part dependent on PKC activation. Even though EGF and PDBu enhanced CCE, the augmentation of this response was greater after exposure to PDBu. Such a difference suggests that other signaling components besides PKC mediated EGF-induced increases in plasma membrane Ca²⁺ influx. This assumption was in accordance with a number of previous studies in which EGF-induced cell signaling was found to entail the activation of myriad interacting pathways.^{4 5 6 7 8 9 10 11 28}

Specific roles of four PKC isoforms in inducing CCE were estimated based on the rank order of inhibition: rottlerin (PKC), hispidin (PKCß), HBDDE (PKC), and PKC V1–2 (PKC) of the enhanced CCE responses resulting from exposure to EGF or PDBu. Figure 2A shows that EGF-induced augmentation of CCE was most effectively inhibited with either rottlerin or hispidin, suggesting that both PKC and PKCß preferentially mediate this enhanced response. On the other hand, after direct stimulation of CCE through PKC activation (Fig. 2B) , rottlerin had a larger inhibitory effect than any of the other three inhibitors that only partially suppressed this response. This difference in isoform mediation of CCE augmentation may be reflective of other cell signaling mediators besides PKC affecting the regulation of CCE.

PKC activation was assessed based on changes in subcellular localization of the isoform of interest. To make this assessment, the tissue was partitioned into M-, C-, Ck-, and N-containing fractions. In each of them, the time-dependent changes were monitored in their specific PKC isoform content. Figures 3 and 4 show that after exposure to EGF and PDBu, the PKC content of the M fraction was preferentially enriched in PKCßI and PKC, whereas there was a corresponding decline in either the C, N, or Ck. These changes could contribute to EGF- and PDBu-induced CCE stimulation because there was a similarity between the time required to detect maximum stimulation of CCE and the onset of translocation of either PKCßI or PKC to the M fraction. During exposure to EGF, the Ck fraction became enriched in PKCßI and PKCßII, whereas exposure to PDBu caused this fraction instead to enrich itself with PKC and PKCßI. PDBu also induced increases in the N fraction of PKC and PKCßI. Based on the results shown in Figures 3 and 4 , it is evident that the profiles of the EGF- and PDBu-induced increases in the PKC content of the M fraction are similar to one another. In both cases, PKC was preferentially translocated to this fraction. This preferential translocation response is similar to that reported in rat liver epithelial (WB) cells.²⁰ Interestingly, there is a correspondence between the efficacy of the PKC inhibitors to suppress EGF-augmented CCE and the preferential translocation of PKC to the M-containing fraction. In other words, rottlerin had the largest inhibitory effect on CCE, which agrees with the finding that PKC translocation to the M fraction was greater than that of any other isoform. Agreement was also observed between the effects of PKC inhibitors on PDBu-stimulated CCE and the fold increase in PKC isoform content of the M fraction. Figure 2 shows that rottlerin had the highest efficacy and that PDBu preferentially induced more PKC translocation to the M fraction (see Fig. 4 ). This agreement in both cases suggests that PKC may be required for mediating EGF-induced increases in plasma membrane Ca²⁺ influx. However, the identity of the plasma membrane substrate undergoing phosphorylation by this isoform is unclear.

Sole reliance on inhibitors to identity PKC isoform specificity in inducing a response may be ambiguous because drug selectivity may be unclear. To circumvent this problem, siRNA technology was used for selective suppression PKCß and PKC protein expression. Two different approaches were used to evaluate the stimulatory effects of EGF and PDBu on CCE after their expression knockdown. Figure 6A indicates that in both types of cells, the EGF-induced stimulation of CCE was fully inhibited to the level obtained after PKC downregulation. Similarly, Figure 6B shows that in these two cell types, PDBu augmentation of CCE was also eliminated. With the second approach to evaluate the stimulation of CCE by EGF and PDBu, changes were measured in Mn²⁺ quench rates induced by EGF and PDBu (see Fig. 7 ). As with the effect of EGF on Ca²⁺ transients (Fig. 6A) , CCE induced by EGF was most fully suppressed after PKCß knockdown, whereas PKC knockdown partially suppressed such increases in Mn²⁺ quench. With PDBu, Mn²⁺ quench was more suppressed in cells transfected with PKC siRNA than with PKCß siRNA. This rank order is in agreement with the results shown in Figure 6B in which PDBu augmentation of CCE was more inhibited by knockdown of PKC than by PKCß. These results suggest that PKCß and PKC are key players in mediating CCE. This rank order is in slight disagreement with the assignment based on the use of inhibitors and measurements of PKC translocation. Nevertheless, there was general agreement between the three approaches in that both PKC and PKCß appeared to be more important than the other isoforms in mediating enhancement of CCE.

The effort to identify which PKC isoforms are preferentially translocated to the plasma membrane has possible clinical significance in optimizing cytokine-induced increases in corneal epithelial wound healing rates. This is conceivable because EGF-induced increases in plasma membrane Ca²⁺ influx are essential for optimizing a mitogenic response to this growth factor.¹⁴ Such a relationship is apparent because siRNA knockdown of TRPC4 protein expression diminished the mitogenic response to this growth factor. This realization, coupled with findings of the present study, suggests that selective stimulation of PKC isoform translocation may augment a mitogenic response to EGF by enhancing the effect of this growth factor on Ca²⁺ influx. Our finding that PKC is one of the four isoforms translocated to the plasma membrane is in agreement with a study in HCECs in which it was shown that EGF induces the translocation of PKC fused to GFP to the plasma membrane.⁴ However, this study did not describe the effect of EGF on plasma membrane translocation of any of the other PKC isoforms. We cannot explain why, in another study, ¹⁷ EGF-induced PKC translocation was undetectable in four subcellular fractions. The present study suggests that PKCßI and PKC may be appropriate drug targets for optimizing EGF-induced increases in plasma membrane Ca²⁺ influx and mitogenesis. This is plausible because there is a close correspondence between the rank order of EGF-induced PKC isoform plasma membrane translocation and the augmentation of CCE.

Different types of plasma membrane calcium influx can be activated, leading to CCE. One type of pathway is SOCs, which were described in HCECs and are also activated by EGF.¹⁴ In human mesangial cells, SOC activation by EGF is mediated through the stimulation of PKC.^{15 29} A component of these pathways is a member of the transient receptor potential (TRP) protein superfamily. In endothelial cells, TRPC1 is a component of SOC and is regulated by PKC.³⁰ One TRP isoform we identified is TRPC4, whose knockdown blunted the mitogenic response to EGF.¹⁴ To obtain insight into how EGF stimulates SOC activity, we determined whether the pan-PKC inhibitor, calphostin C, suppressed this response. Figure 8E provides validation that EGF stimulates SOC activity because the SOC-originated current was enhanced approximately twofold when the cell was voltage clamped at –120 mV. Such EGF-stimulated currents were completely suppressed after the inhibition of PKC activity with 1 µM calphostin C. Even though PKC activation is a component of the pathway mediating EGF stimulation of SOC, it is unclear whether this response was caused by direct phosphorylation by PKC of TRPC4. There is some suggestion in another recent study using COS7 cells that phosphorylation of this substrate could mediate EGF stimulation of SOC activity. In this report, it was shown that tyrosine phosphorylation of TRPC4 by Src family tyrosine kinases (STKs) rather than PKC affects such control.¹⁶ On the other hand, SOC activation in prostate cancer epithelial cells is dependent on TRPC4 interaction with a calcium influx factor.³¹ It is conceivable in HCECs that SOC activation by EGF in HCECs also could ultimately be mediated by tyrosine phosphorylation through STK.

In summary, EGF-induced stimulation of CCE and SOC activity is dependent on selective increases in the activity of different PKC isoforms. It is unclear whether such increases directly account for the phosphorylation events leading to increases in SOC activity. Nevertheless, EGF augmentation of CCE is preferentially dependent on the stimulation of PKCß and PKC, whereas stimulation of this response by PDBu appears to be mediated more through increases in PKC rather than PKCß activity. This difference could reflect the fact that increases in SOC activity can be modulated by many cell-signaling pathways linked to EGF receptor stimulation. In this network, the participating PKC isoforms may not be the same as those mobilized by direct stimulation of PKC with PDBu.

	Acknowledgements

 
The authors thank Uwe Pleyer for encouragement, guidance, and helpful discussions and Dennis Wong for assistance with the experiments.

	Footnotes

 
Supported by National Institutes of Health Grant EY04795.

Submitted for publication March 10, 2006; revised April 9 and May 1, 2006; accepted July 3, 2006.

Disclosure: F. Zhang, None; Q. Wen, None; S. Mergler, None; H. Yang, None; Z. Wang, None; V.N. Bildin, None; P.S. Reinach, None

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

Corresponding author: Peter S. Reinach, Department of Biological Sciences, College of Optometry, State University of New York, 33 West 42nd Street, New York, NY 10036; preinach{at}sunyopt.edu.

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