March 2003
Volume 44, Issue 3
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Lens  |   March 2003
IGF-I-Induced Phosphorylation of Connexin 43 by PKCγ: Regulation of Gap Junctions in Rabbit Lens Epithelial Cells
Author Affiliations
  • Dingbo Lin
    From the Department of Biochemistry and
  • Daniel L. Boyle
    Division of Biology, Kansas State University, Manhattan, Kansas.
  • Dolores J. Takemoto
    From the Department of Biochemistry and
Investigative Ophthalmology & Visual Science March 2003, Vol.44, 1160-1168. doi:10.1167/iovs.02-0737
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      Dingbo Lin, Daniel L. Boyle, Dolores J. Takemoto; IGF-I-Induced Phosphorylation of Connexin 43 by PKCγ: Regulation of Gap Junctions in Rabbit Lens Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2003;44(3):1160-1168. doi: 10.1167/iovs.02-0737.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

purpose. To determine the role of PKCγ in insulin-like growth factor (IGF)-I-induced phosphorylation of connexin (Cx)43 and control of gap junctions in lens epithelial cells.

methods. N/N1003A rabbit lens epithelial cells were used in the experiments. PKC translocation or in vivo Cx43 phosphorylation on serine was determined by Western blot analysis. Gap junction activity was measured by scrape-loading/dye-transfer assay. The number of cell surface gap junction plaques was detected by confocal microscopy. The interaction between PKCγ and Cx43 was determined by coimmunoprecipitation. In vitro Cx43 phosphorylation was assayed by PKC assay kit. Endogenous sn-1,2-diacylglycerol (DAG) was measured by detecting 32P-labeled phosphatidic acid.

results. IGF-I stimulated activation and translocation of PKCγ in a dose- and time-dependent manner, acidic FGF (aFGF) had no effect on translocation of PKCγ, and PKCα was not translocated by IGF-I at 25 ng/mL. PKCγ translocation resulted in coimmunoprecipitation with and phosphorylation of Cx43. IGF-I- or DAG-induced activation of PKCγ caused a decrease in gap junctions. IGF-I increased endogenous DAG. Exogenous CaCl2 and DAG stimulated PKCγ translocation. TMB-8, an internal calcium mobilization inhibitor, blocked CaCl2-induced PKCγ translocation; however, it had no effect on IGF-I- or DAG-induced translocation of PKCγ.

conclusions. PKCγ mediated IGF-I-induced decreases in gap junctional communication through interaction with and phosphorylation of Cx43. IGF-I caused an increase in DAG, and this increased translocation of PKCγ, whereas mobilization of calcium was not essential for IGF-I-stimulated translocation of PKCγ.

Gap junctions are nonspecific channels between adjacent cells that allow the passage of small (<1 kDa) molecules, metabolites, and ions. A gap junction channel is formed by two connexons contributed equally by adjacent cells. A connexon is an assembly of six connexin 43 (Cx43) proteins in the lens epithelial cells, whereas Cx46 and Cx50 are found in the lens fiber cells. 1 2 Reports have indicated that gap junctions between the lens cells play a crucial role in the intercellular metabolic support essential for lens survival. 2 3 Studies in knockout mice have demonstrated that gap junctions are important for maintaining lens transparency. When the Cx46 gene was knocked out, the mutant mice showed development of nuclear opacity within 3 weeks of birth. 4 5 6  
Gap junctions are regulated by many environmental factors, such as stress factors, drugs, and growth factors. Previous studies have reported that diabetes causes a decrease in gap junction activity in the lens. 7 In our laboratory, we have reported that diabetes or galactosemia causes a downregulation of protein kinase C (PKC)γ activity and gap junction activity. 8 9 The long-term goal of our study was to understand the involvement of PKCγ in diabetic cataractogenesis. In this study, the role of insulin-like growth factor (IGF)-I in control of PKCγ was determined. 
IGF-I is a potent growth factor that acts in endocrine, autocrine, and paracrine settings to regulate cell proliferation, differentiation, metabolism, and apoptosis. 10 11 12 IGF-I actions are mediated by IGF-I receptors in various types of mammalian cells. 13 Recent studies have provided evidence that IGF-I stimulates influx of calcium and production of diacylglycerol. 14 15 16 IGF-I stimulation promotes nuclear production of DAG and nuclear translocation of PKCα in Swiss 3T3 mouse fibroblasts cells. 17 IGF-I is known to activate IGF-I receptors in oocytes, thus triggering a cascade that involves the activation of MAPK pathways. 18 In vitro studies have shown that MAPK can phosphorylate Cx43 at serines 255, 279, and 282. 19  
PKCγ, a type of conventional PKC isozyme, has been identified as an inhibitor of gap junction activities in the lens epithelial cells through its phosphorylation of Cx43. 1 9 As far as we are aware, no report has been focused on the function of PKCγ in IGF-I-mediated regulation of gap junctional communication in the lens. In the current study, IGF-I activated PKCγ, which, in turn, increased the binding of Cx43 to PKCγ and phosphorylation of Cx43 by PKCγ, resulting in disassembly of gap junction plaques and decreased gap junction activity. DAG may work as a second messenger in signaling of PKCγ activation by IGF-I, whereas mobilization of calcium does not seem to be the primary signal for PKCγ. 
Material and Methods
Cell Culture
N/N1003A rabbit lens epithelial cells were cultured in 75-cm2 flasks in Dulbecco’s modified Eagle’s medium (DMEM, low glucose; Invitrogen, San Diego, CA) supplemented with 10% fetal bovine serum and 50 μg/mL gentamicin, 0.05 U/mL penicillin, and 50 μg/mL streptomycin (pH 7.4) at 37°C in an atmosphere of 90% air and 10% CO2. The cells were used when they reached 90% confluence. 
Western Blot Analyses
Western blot analyses were conducted as described previously. 1 Briefly, cells were homogenized with 50 mM Tris-HCl (pH 7.5), 20 mM MgCl2, 25 μg/mL aprotinin, 25 μg/mL leupeptin, and 1 mM phenylmethylsulfonyl fluoride (PMSF). Soluble and membrane fractions were separated by ultracentrifugation at 100,000g for 1 hour at 4°C. Ten micrograms total protein was separated by 7.5% SDS-PAGE, transferred to nitrocellulose membranes, and probed with antibodies against PKCα or -γ (1:1000). The antibodies for PKCα and -γ were from Transduction Laboratories (Lexington, KY), and were monoclonal antibodies produced with specific peptides used as immunogens. They are specific for either PKCα or -γ. Anti-Cx43 were from Chemicon (Temecula, CA). The immunogen of anti-Cx43 is the peptide corresponding to positions 252 to 270 of the mouse Cx43 sequence. This monoclonal Cx43 antibody reacts with only a single 43-kDa band on SDS-PAGE. Immunoreactive bands were detected by chemiluminescence (ECL; Pierce, Rockford, IL). The bands were digitized by computer (UN-Scan-It software; Silk Scientific, Orem, UT). 
Coimmunoprecipitation
Ninty percent confluent cells in a 75-cm2 flask were collected and lysed on ice with 1 mL of lysis buffer followed by homogenization and sonication. The cell lysis buffer contains 20 mM Tris-HCl (pH 7.5), 0.5 mM EDTA, 0.5 mM EGTA, 0.5% Triton X-100, 25 μg/mL aprotinin, and 25 μg/mL leupeptin. After centrifugation at 13,000 rpm (20,000g) for 20 minutes, the supernatants were collected and used as whole-cell extracts. Immunoprecipitation antibodies (5 μg/mL) against PKCγ or Cx43 (1:1000; Chemicon) were added to 1 mL whole-cell extracts containing 3 μg/μL total protein, and incubated at 4°C overnight with constant rotation. After this, 20 μL protein-agarose beads (A/G Plus; Santa Cruz Biotechnology, Santa Cruz, CA) were added to the mixture and were further incubated for another 2 hours. The beads were collected by centrifugation, washed with phosphate-buffered saline (PBS) four times, extracted with 20 μL 1× sample loading buffer (containing 50 mM Tris-HCl [pH 6.8], 100 mM dithiothreitol [DTT], 2% sodium dodecyl sulfate [SDS], 10% glycerol, and 0.1% bromophenol blue), and boiled for 3 minutes. Western blot analyses were used to visualize immunoreactive bands, as described earlier. 
In Vitro and In Vivo Phosphorylation of Cx43 by PKCγ
N/N1003A lens epithelial cells were treated with or without 25 ng/mL IGF-I and harvested at different time intervals. Primary PKCγ antibodies (5 μg) were added to 1 mL of the whole-cell extracts containing 3 μg/μL total cell proteins at 4°C overnight with constant rotation. After this, 20 μL protein-agarose beads were added to the mixture and incubated for another 2 hours. The bead-protein complexes were used for an in vitro phosphorylation assay according to the kit instructions (Protein Kinase C Assay; Calbiochem, La Jolla, CA). To start the PKC phosphorylation assay, 5× PKC substrate solution (250 μM Ac-MBP [residues 4–14], 100 μM adenosine triphosphate [ATP], 5 mM CaCl2, 100 mM MgCl2, 20 mM Tris [pH 7.5]) or the 5× inhibitor solution (100 μM PKC [residues 19–36], and 20 mM Tris [pH 7.5]), a blank control, not for the PKC inhibition test, was added. To begin the reaction, a PKC reaction mixture consisting of 25 Ci/mL [γ-32P] ATP was added to the bead-protein complexes, gently pipetted, and incubated at 30°C for 5 minutes. The reaction was stopped by adding 20 μL Tris-glycine/SDS sample buffer, and the samples were boiled for 5 minutes. Proteins were separated by 7.5% SDS-PAGE, and the gel was scanned by a commercial system (Typhoon PhosphoImage System; Amersham Pharmacia Biotech, Piscataway, NJ). Phospho-Cx43 bands were imaged, digitized, and graphed. 
For in vivo phosphorylation assays, the bead-protein complexes prepared were mixed and boiled with protein loading buffer and subjected to SDS-PAGE (12.5%) directly. The separated proteins were transferred to nitrocellulose membranes and probed with anti-phosphoserine antibodies (1:500; Chemicon) to detect the relative abundance of phosphoserine in Cx43. 
Confocal Microscopy Gap Junction Assays
Cell surface gap junction Cx43 plaque assays were performed as described previously. 1 Cell gap junction activities were determined by scrape-loading/dye-transfer (SL/DT) assay. 9 20 N/N1003A lens epithelial cells were grown on glass coverslips in six-well plates. When cells reached 90% confluence, 2.5 μL 1% lucifer yellow and 2.5 μL 1% rhodamine dextran (Molecular Probes, Madison, WI) were added at the center of coverslips where a cut had been made across the cell surfaces. The cells were incubated for 1 minute at room temperature and washed three times with PBS and incubated with normal medium for 10 minutes, to allow the dye to transfer. Cells were fixed with 2.5% paraformaldehyde for 10 minutes, and the efficiency of dye transfer was monitored by confocal microscopy. 9 20 Gap junction activity was expressed as number of cells transferred per area ± SD. 
Endogeneous DAG Assay
Sample preparation and radioenzymatic assays were performed according to the instructions for the DAG assay kit (Amersham Pharmacia Biotech). Total lipids were extracted with chloroform-methanol and used as the substrates for DAG kinase. Phosphatidic acid (PA) which was labeled by 32P was produced by the reaction. The product mixtures were separated by thin-layer chromatography, and zones corresponding to 32P PA were visualized by submitting the dried plates to autoradiography overnight. The spots containing 32P PA were quantitated by scintillation counting. Endo-DAG levels were calculated from DAG standard curves. 
Results
Translocation of PKCγ Stimulated by IGF I
In many types of mammalian cells, PKC is cytoplasmic and inactive. PKC translocates from the cytosol to the membrane once it is activated by activators, such as calcium, DAG, phorbol ester (12-O-tetradecanoylphorbol 13-acetate; TPA) or growth factors. PKCγ has been reported to regulate gap junctional communication in the lens. 1 However, the function of growth factors such as IGF-I on activation of lens PKCγ has never been reported. When the lens epithelial cells were treated with IGF-I, translocation of PKCγ, but not of PKCα, occurred in a dose-dependent manner (Fig. 1A , top). No PKCγ translocation occurred when the cells were treated with acidic FGF (Fig. 1A , bottom). Quantitative analyses for translocation of PKCγ by IGF-I are presented in Figure 1C . IGF-I (1 ng/mL) stimulated the translocation of PKCγ 20 minutes after treatment, and maximum efficiency was reached when the cells were exposed to 25 ng/mL IGF-I for 20 minutes (Fig. 1C , top). When 25 ng/mL of IGF-I was applied to the cells, PKCγ was translocated from the cytosol to the membranes as early as 10 minutes after treatment (Figs. 1B 1C, bottom). 
Enhancement of the Interaction between PKCγ and Cx43
Coimmunoprecipitation assays were used to analyze the possible interactions between PKCγ and Cx43, the major gap junction protein in the lens epithelial cells, after treatment with IGF-I. For equal loading, 3 mg of the total cell protein of the whole-cell extracts were incubated with 5 μg of antibodies against PKCγ or Cx43. Figure 2A shows that coimmunoprecipitation of PKCγ and Cx43 was increased by 4-to 10-fold when the cells were treated with 25 ng/mL IGF-I for 30 minutes, according to the quantitation analysis of Western blot bands (Fig. 2A , bottom). As a control, anti-PKCγ and anti-Cx43 antibodies were used as the probes to hybridize the blots shown in Figures 2B and 2C , respectively. The results indicated that coimmunoprecipitation of PKCγ with Cx43 was specific. 
Enhancement of Phosphorylation of Cx43 by PKCγ
Connexins are phosphoproteins, and their phosphorylation status contributes to the regulation of assembly and disassembly of gap junction channels. 1 21 22 23 24 Previous reports showed that PKC isozymes phosphorylate serine residues at the C terminus of Cx43. 1 21 23 24 The role of IGF-I in the activities of PKCγ and phosphorylation of Cx43 was determined. 
Equal amounts of IGF-I-treated whole-cell extracts were used for coimmunoprecipitation of Cx43 with the PKCγ antibody (described earlier). The bead-protein complexes were used for in vitro PKC assays. The phosphorylation patterns were imaged, and the quantitative analyses (of three samples ± SD) are presented in Figure 3A . We found multiple PKCγ-interactive proteins when we used anti-PKCγ to precipitate its binding proteins. For example, 14-3-3 adapter proteins also interacted with PKCγ (data not shown). We probed the blot with monoclonal anti-Cx43 and found the specific band located on the blot at approximately 43 kDa. The results demonstrate that 25 ng/mL of IGF-I caused increased phosphorylation of Cx43 and that the phosphorylation levels reached maximum at 20 minutes and returned to control levels at 45 minutes, suggesting that IGF-I is a transient growth factor signal. This suggests activation of PKCγ enzyme activity by IGF-I. 
Cx43 is also phosphorylated at threonine and tyrosine. However, the phorbol ester-induced changes in gap junctions resulted only in changes in serine phosphorylation. Therefore, in the current study, we used anti-phosphoserine antisera as a probe to analyze the in vivo total phosphorylation of Cx43 at serine residues (Fig. 3B) . Whole-cell extracts from IGF-I-treated cells were obtained at different time intervals. Equal whole-cell extracts were immunoprecipitated with 5 μg anti-Cx43 antibody, subjected to Western blot analysis, and probed by anti-phosphoserine. In Figure 3B only the Cx43 band is shown. However, phosphorylated PKCγ and IgG bands are also present on this gel. Cx43 and p-Cx43 have almost identical mobilities on 12.5% SDS-PAGE. The results demonstrated that phosphorylation of serine residues of Cx43 was increased in a time-dependent manner after treatment with IGF-I (Fig. 3B) . Equal loading was verified in the stripped blot probed with anti-Cx43 (Fig. 3C)
These data (Figs. 3A 3B) collected from both in vitro and in vivo phosphorylation studies indicated that in vitro maximal phosphorylation of Cx43 occurred at 20 minutes, but significant increases in phosphoserine were detected only at 30 minutes. Fig 3A shows newly 32P labeled phospho-Cx43 levels by PKCγ initiated by addition of ATP. Fig 3B shows the accumulation of phosphoserine on Cx43 during IGF-I treatment in live cells and is the result of kinase and phosphatase activity. It may, therefore, take more time to observe a change in phosphoserine in Cx43 in vivo. 
Effect of IGF-I on Cell Surface Gap Junctional Communication
Previous publications have documented that phosphorylation of Cx43 results in disassembly of gap junction channels and that the activation of PKCγ decreases the gap junctional communications through phosphorylation of Cx43 in lens epithelial cells. 1 In the current study, IGF-I activated PKCγ to translocate to membranes, interact with and phosphorylate Cx43, and decrease gap junction activity. We determined both the numbers of cell surface gap junction plaques and gap junction activity by confocal microscopy. IGF-I (25 ng/mL) was added to treat N/N1003A cells for 0, 10, 20, and 30 minutes. The cells, fixed and stained with anti-PKCγ antibody (green) and anti-Connexin 43 antibody (red), were viewed under a laser scanning confocal microscope (Carl Zeiss, Thornwood, NY). Examples of the images are shown in Figures 4 and 7 , and the number of plaques per micrometer was determined from similar images. Figure 4A shows that cells treated with IGF-I have reduced cell surface Cx43 plaques at 20 and 30 minutes. Incubation with IGF-I for 30 minutes resulted in a 57.2% decrease in the number of plaques. 
Figure 4B shows the changes in gap junction activities during IGF-I treatments. N/N1003A cells grown in the absence of IGF-I had a gap junction dye transfer mean of 28.8 cells. IGF-I at 25 ng/mL decreased the number of dye labeled cells by 44.6% in a time-dependent manner. The results presented herein indicate that both gap junction activity and the number of cell surface gap junction Cx43 plaques in the lens epithelial cells are decreased by IGF-I. 
The 90% confluent cells were lysed with cell lysis buffer (see the Methods section), and the supernatants were used to measure total Cx43 extracts by Western blot. Twenty micrograms total protein was applied to Western blot and probed with Cx43 antisera. Figure 4C demonstrates that total cell Cx43 did not decrease after incubation with 25 ng/mL IGF-I for up to 30 minutes. This suggests that IGF-I causes internalization of Cx43, not degradation. 
Effects of TMB-8, a Calcium Mobilization Inhibitor, on PKCγ Translocation Stimulated by IGF-I
A growth factor, such as IGF-I, transmits signals by interaction with its receptors, and subsequent activation of phospholipase C, which, in turn, catalyzes the hydrolysis of PIP2 to yield inositol-1,4,5-trisphosphate (IP3) and DAG. IP3 binds to receptors or the endoplasmic reticulum to further open a calcium transport channel, which triggers diverse cellular processes. DAG remains embedded in the plasma membrane to activate PKC. Current research was focused on the probable signaling systems involved in IGF-I-evoked PKCγ activation. We set CaCl2 as a positive control to determine the calcium activation of PKCγ. The dose experiments showed that CaCl2 induced translocation of PKCγ in a dose-dependent manner. Five mM CaCl2 for 25 minutes translocated PKCγ to membranes (Fig. 5A , left), whereas total translocation was observed at 25 mM (Fig. 5A , top). The results obtained from time-course studies revealed that translocation of PKCγ to membranes was initiated in less than 5 minutes when the cells were treated with 25 mM CaCl2 (Fig. 5A , bottom). Application of 10 to 35 μM TMB-8, an internal inhibitor of calcium mobilization, to N/N1003A cells for 15 minutes before 10 mM Ca+2 treatment partially abolished the effect of CaCl2 on translocation of PKCγ (Fig. 5B , lanes 5–7). However, TMB-8 at 10 or 35 μM did not affect the PKCγ translocation stimulated by 25 ng/mL IGF-I (Fig. 5B , lanes 8–10) or 1 μM DAG (Fig. 5B , lanes 11–13). This suggests that internal calcium mobilization is not a predominant effector of PKCγ translocation initiated by IGF-I or DAG. 
DAG-Activated PKCγ
DAG is considered as an activator of conventional PKCs in mammalian cells. Exogenous DAG was applied to N/N1003A cells for 30 minutes at different doses, and the Western blot profiles are shown in Figure 6A . PKCγ and -α, two predominant conventional PKC isozymes in rabbit lens epithelial cells, translocated to the membranes in a dose-dependent and time-dependent manner after treatment with DAG. Previous reports have demonstrated that the K d for DAG binding differs between PKCγ and -α, with the former being activated by 10-fold lower doses. 25 26 Thus, PKCγ was also more sensitive to DAG levels, because substantial translocation of PKCγ at 0.5 μM DAG versus a PKCα dose of 2.5 μM (Fig. 6A) was observed. DAG (1 μM) causes translocation of PKCγ by 5 minutes (Fig. 6A , bottom). When the cells were pretreated with TMB-8 for 15 minutes followed by 1 μM DAG for 30 minutes, there was no detectable effect of TMB-8 on the translocation of PKCγ (Fig. 5B , lanes 11–13). This suggests that DAG initiates translocation of PKCγ, independently of calcium mobilization. 
To determine whether IGF-I regulates PKCγ through DAG, we analyzed the changes of endogenous DAG levels before and after IGF-I treatments. The results in Figure 6B demonstrate that cellular DAG levels increased up to 146% compared with the control, after treatment with 25 ng/mL of IGF-I for 20 minutes, and that prolonged treatment showed DAG levels returning to baseline levels at 30 minutes. The increase in the endogenous DAG level stimulated by 1 μM exogenous DAG treatment is similar to that obtained with 25 ng/mL IGF-I (data not shown). Optimal time (15 minutes) with 25 ng/mL IGF-I raised cellular DAG levels to approximately 200 pmol/mg tissue and 1 μM DAG elevated these levels to approximately 300 pmol/mg tissue (data not shown). The increased cellular DAG induced by IGF-I is in the same range as that induced by 1 μM DAG. This level causes translocation of PKCγ and not -α (see Fig. 6A , top). This may account for a preferential activation of PKCγ by IGF-I. 
Effect of DAG on Cell Surface Cx43 Plaques and Gap Junction Activity
DAG was added to N/N1003A cells for 0, 15, and 30 minutes. The cells, fixed and stained with anti-PKCγ antibody (green) and anti-Connexin 43 antibody (red), were viewed under a confocal microscope, and the images were obtained. Figure 7A shows that cells treated with 1 μM DAG have reduced cell surface Cx43 plaques. Incubation with 1 μM DAG for 30 minutes resulted in a 59.4% decrease in cell surface Cx43 plaques. 
After N/N1003A cells were treated with or without 1 μM of DAG for 15 or 30 minutes, the cells were used for SL/DT assays to evaluate the gap junction activity. Figure 7B demonstrates the decreases in gap junction activities during DAG treatments. DAG at 1 μM for 30 minutes decreased the value of dye-labeled cells by 42.9% in a time-dependent manner, as shown in the example images. The results presented herein indicate that both gap junction activity and the number of cell surface gap junction Cx43 plaques in the lens epithelial cells are decreased by DAG. 
Total cell Cx43 levels were determined by Western blot probed with Cx43 antisera. No decrease in total cell Cx43 level was detected in DAG-activated cells (data not shown). 
Discussion
Diabetes is the third leading cause of death in the United States after heart disease and cancer. Experiments have demonstrated that during diabetes and galactosemia, lens PKCγ levels are dramatically decreased. 8 9 In myocardial tissue, DAG levels are elevated during diabetes, and this increases PKCγ activity and decreased gap junction activity. 27 28 29 However, it is still unclear how PKCγ functions in the lens cell-cell gap junction communication. In the present study, we used lens epithelial cells in culture as our experimental model to investigate the regulation of PKCγ and gap junctions by IGF-I. We find that IGF-I can cause PKCγ translocation, and PKCγ subsequently interacts with and phosphorylates Cx43. IGF-I reduced gap junction activity and the numbers of cell surface gap junction plaques. The results further suggest that diacylglycerol is the messenger for activation of PKCγ by IGF-I. 
IGF-I is an important growth factor involved in diverse growth and differentiation effects. IGF-I is transported to the eyes through blood and then secreted into the ocular fluids. 30 IGF-I transduces its signals through activation of its receptors located in plasma membrane of the lens epithelial cells, which triggers two major signaling cascades, the MAPK and PI 3-kinase pathways. 30 31 32 Klok et al. 30 reported that 50 ng/mL IGF-I is sufficient to maintain features of rat lens epithelium differentiation once differentiation is initiated by FGF-2. Similar with other IGF-I dose treatments in lens, 30 10 to 25 ng/mL IGF-I can regulate translocation of PKCγ and gap junctions in N/N1003A cells, indicating that IGF-I signaling may occur through activation of PKCγ. 
The potential downstream signal molecules, such as calcium and DAG, in the signaling of IGF-I and activation of PKCγ in N/N1003A cells were investigated. TMB-8, an inhibitor for internal calcium mobilization, does not abolish the translocation of PKCγ stimulated by IGF-I, suggesting that IGF-I activation of PKCγ is less sensitive to calcium mobilization. Calcium influx may be the principal force for PKCγ membrane-localization induced by Ca2+. 13 33 Similar experiments have been performed using GFP-tagged C2 domains and demonstrating that calcium mobilization is essential for transient membrane localization and calcium influx is necessary for persistent localization. 33  
Both PKCα and -γ have two distinct lipid DAG-binding domains, rich in cysteine, referred to as C1A and C1B domains and occurring within the C1 domain. 34 C1 domain DAG-binding affinities differ between PKCα and -γ. Compared with PKCγ, PKCα has lower DAG-binding affinity. 26 34 C1A domains provide the interaction with membrane lipid residues, whereas the C1B domains are interaction sites for DAG. 34 35 36 Elegant studies using GFP-tagged C1A and C1B domains and high- or low-frequency calcium spikes indicate that DAG binding to C1A prolongs the response to high-calcium binding at C1B sites. 36 In our lens epithelial cells, IGF-I yielded DAG, which may not only increase the membrane affinity for PKCγ, but may also activate PKCγ, but not PKCα, by directly binding to the C1B domain of PKCγ. It can be hypothesized that the lens epithelial cell PKCγ has a DAG sensing threshold much lower than that observed for PKCα. This would allow the different growth factors to activate PKCγ without activation of PKCα. 
Gap junction channels allow small signaling molecules, metabolites, and ions to pass through to regulate cellular homeostasis and cell-cell signaling. 37 We have shown that PKCγ inhibits gap junction activity in the lens epithelial cells through interaction with and phosphorylation of Cx43 on serine residues. 1 9 Cx43 phosphorylation on multiple serine and tyrosine residues contributes to the alteration of gap junction activity. 38 39 Our research demonstrates that activation of PKCγ by IGF-I subsequently results in the decrease of gap junction activity through PKCγ phosphorylation of Cx43. The PKCγ-induced decrease in cell surface Cx43 is not a result of Cx43 degradation. At present the internalization and processing of Cx43 is being studied. 
Control of gap junctions could be critical to cell growth and to responses of cells to stress. Our previous study reported that PKCα and -γ have opposite effects on control of gap junctions. Activation of PKCα results in increases in cell surface gap junctions, whereas activation of PKCγ causes a decrease in gap junctions. 1 The switch between assembly and disassembly of gap junctions by PKCα and -γ can function physiologically when the cells are exposed to environmental factors, such as IGF-I, EGF, or stress factors. 
In summary, IGF-I signaling in the lens epithelial cells may occur through binding to IGF-I receptors, which, in turn, triggers production of DAG through PLCs. PKCγ then binds to DAG, translocates to membranes, and further phosphorylates Cx43, which results in disassembly of gap junctions. 
 
Figure 1.
 
IGF-I stimulated translocation of PKCγ, but not of PKCα. (A) N/N1003A cells were treated with 0, 1, 5, 10, 25, and 50 ng/mL IGF-I after 20 minutes of incubation. Proteins (10 μg) of membrane or supernatant fractions were separated on 7.5% SDS-PAGE and Western blot analysis performed with antibodies against PKCα or -γ. IGF-I induced translocation of PKCγ, but not of PKCα, to the membrane fractions. Cells were treated with acidic FGF (aFGF) as a negative control to demonstrate no translocation of PKCγ or -α. (B) The N/N1003A cells were treated with or without 25 ng/mL IGF-I for 0, 5, 10, 20, or 30 minutes. The supernatant and membrane fractions were prepared and Western blots were probed with PKCγ antibody. The cells were collected at each time interval, separated into supernatants and membrane fractions, and subjected to Western blot analysis to detect the translocation of PKCγ. Time courses of PKCγ translocation are shown. (C) Quantitative analysis of results in three experiments (±SD), showing PKCγ translocation in a dose- and time- dependent manner.
Figure 1.
 
IGF-I stimulated translocation of PKCγ, but not of PKCα. (A) N/N1003A cells were treated with 0, 1, 5, 10, 25, and 50 ng/mL IGF-I after 20 minutes of incubation. Proteins (10 μg) of membrane or supernatant fractions were separated on 7.5% SDS-PAGE and Western blot analysis performed with antibodies against PKCα or -γ. IGF-I induced translocation of PKCγ, but not of PKCα, to the membrane fractions. Cells were treated with acidic FGF (aFGF) as a negative control to demonstrate no translocation of PKCγ or -α. (B) The N/N1003A cells were treated with or without 25 ng/mL IGF-I for 0, 5, 10, 20, or 30 minutes. The supernatant and membrane fractions were prepared and Western blots were probed with PKCγ antibody. The cells were collected at each time interval, separated into supernatants and membrane fractions, and subjected to Western blot analysis to detect the translocation of PKCγ. Time courses of PKCγ translocation are shown. (C) Quantitative analysis of results in three experiments (±SD), showing PKCγ translocation in a dose- and time- dependent manner.
Figure 2.
 
Interaction between PKCγ and Cx43 was enhanced by IGF-I. (A) N/N1003A cells were harvested with or without treatment of 25 ng/mL of IGF-I for 30 minutes. The whole-cell lysates (3 mg protein) were incubated with 5 μg PKCγ antibody or Cx43 antibody at 4°C overnight. Top: Coimmunoprecipitation complexes were subjected to Western blot with anti-Cx43 or anti-PKCγ used as probes. Bottom: Quantitative analysis of results in three experiments (±SD), showing IGF-I-enhanced coimmunoprecipitation of PKCγ and Cx43. IP, immunoprecipitation; IB, immunoblot. (B) Negative controls for coimmunoprecipitation. Lane 1: anti-PKCγ antisera+whole-cell extracts (3 mg protein)+protein-agarose beads; lane 2: whole-cell extracts+protein-agarose beads; lane 3: protein-agarose beads; lane 4: nonimmunoprecipitated whole-cell extracts (10 μg); lane 5: anti-PKCγ antisera+proteinagarose beads. For Western blot, anti-PKCγ was used as a primary antibody and anti-mouse IgG as a secondary antibody, and results were visualized with chemiluminescence. (C) The negative control for coimmunoprecipitation. Lane 1: anti-Cx43 antisera+whole-cell extracts (3 mg protein)+protein-agarose beads; lane 2: nonimmunoprecipitated whole-cell extracts (10 μg); lane 3: whole-cell extracts+protein-agarose beads; lane 4: protein-agarose beads; lane 5: anti-Cx43 antisera+protein-agarose beads. For Western blot, anti-Cx43 was used as a primary antibody and anti-rabbit IgG as a secondary antibody, with results visualized with chemiluminescence.
Figure 2.
 
Interaction between PKCγ and Cx43 was enhanced by IGF-I. (A) N/N1003A cells were harvested with or without treatment of 25 ng/mL of IGF-I for 30 minutes. The whole-cell lysates (3 mg protein) were incubated with 5 μg PKCγ antibody or Cx43 antibody at 4°C overnight. Top: Coimmunoprecipitation complexes were subjected to Western blot with anti-Cx43 or anti-PKCγ used as probes. Bottom: Quantitative analysis of results in three experiments (±SD), showing IGF-I-enhanced coimmunoprecipitation of PKCγ and Cx43. IP, immunoprecipitation; IB, immunoblot. (B) Negative controls for coimmunoprecipitation. Lane 1: anti-PKCγ antisera+whole-cell extracts (3 mg protein)+protein-agarose beads; lane 2: whole-cell extracts+protein-agarose beads; lane 3: protein-agarose beads; lane 4: nonimmunoprecipitated whole-cell extracts (10 μg); lane 5: anti-PKCγ antisera+proteinagarose beads. For Western blot, anti-PKCγ was used as a primary antibody and anti-mouse IgG as a secondary antibody, and results were visualized with chemiluminescence. (C) The negative control for coimmunoprecipitation. Lane 1: anti-Cx43 antisera+whole-cell extracts (3 mg protein)+protein-agarose beads; lane 2: nonimmunoprecipitated whole-cell extracts (10 μg); lane 3: whole-cell extracts+protein-agarose beads; lane 4: protein-agarose beads; lane 5: anti-Cx43 antisera+protein-agarose beads. For Western blot, anti-Cx43 was used as a primary antibody and anti-rabbit IgG as a secondary antibody, with results visualized with chemiluminescence.
Figure 3.
 
Cx43 phosphorylation was enhanced by IGF-I. (A) In vitro phosphorylation of Cx43. N/N1003A cells were treated with 25 ng/mL IGF-I and harvested at the time intervals shown. Primary antibodies (5 μg) were added to 1 mL of whole-cell extracts containing 3 μg/μL total proteins for immunoprecipitation. Top: the protein complexes were used for in vitro phosphorylation. Bottom: the results of three experiments ± SD were subjected to quantitative analysis. IGF-I caused enhanced PKCγ activity that resulted in phosphorylation of Cx43. The phosphorylation levels reached maximum by 20 minutes after IGF-I treatment. p-Cx43, 32P-labeled Cx43. (B) In vivo phosphorylation of Cx43. Top: whole-cell Cx43 was immunoprecipitated from samples of N/N1003A cells treated with 25 ng/mL of IGF-I at 0, 10, 20, and 30 minutes. The immunoprecipitated Cx43 was analyzed by 12.5% SDS-PAGE and Western blot, probed with anti-phosphoserine antisera. The cells treated with IGF-I had an increase in serine phosphorylation of Cx43 compared with control cells after 30 minutes. Bottom: mean ± SD of results obtained in three experiments. (C) Top: the blot was then stripped and reprobed with anti-Cx43 to show the equal loading for Cx43. Bottom: mean ± SD of results obtained in three experiments. IP, immunoprecipitation; IB, immunoblot.
Figure 3.
 
Cx43 phosphorylation was enhanced by IGF-I. (A) In vitro phosphorylation of Cx43. N/N1003A cells were treated with 25 ng/mL IGF-I and harvested at the time intervals shown. Primary antibodies (5 μg) were added to 1 mL of whole-cell extracts containing 3 μg/μL total proteins for immunoprecipitation. Top: the protein complexes were used for in vitro phosphorylation. Bottom: the results of three experiments ± SD were subjected to quantitative analysis. IGF-I caused enhanced PKCγ activity that resulted in phosphorylation of Cx43. The phosphorylation levels reached maximum by 20 minutes after IGF-I treatment. p-Cx43, 32P-labeled Cx43. (B) In vivo phosphorylation of Cx43. Top: whole-cell Cx43 was immunoprecipitated from samples of N/N1003A cells treated with 25 ng/mL of IGF-I at 0, 10, 20, and 30 minutes. The immunoprecipitated Cx43 was analyzed by 12.5% SDS-PAGE and Western blot, probed with anti-phosphoserine antisera. The cells treated with IGF-I had an increase in serine phosphorylation of Cx43 compared with control cells after 30 minutes. Bottom: mean ± SD of results obtained in three experiments. (C) Top: the blot was then stripped and reprobed with anti-Cx43 to show the equal loading for Cx43. Bottom: mean ± SD of results obtained in three experiments. IP, immunoprecipitation; IB, immunoblot.
Figure 4.
 
IGF-I decreased cell surface gap junction plaques and gap junction activity. (A) IGF-I (25 ng/mL) decreased the number of cell surface Cx43 plaques per square micrometer of the measured area. N/N1003A cells were treated with or without 25 ng/mL of IGF-I for 0, 10, 20, and 30 minutes, and cells were then fixed with 2.5% paraformaldehyde in PBS for 10 minutes, as described previously. 9 Top: sample images from each time point. Bottom: after incubation for 30 minutes with IGF-I, cell surface Cx43 plaques were reduced by 57.2%. Data are the mean ± SD of results in triplicate experiments. The results were statistically significant in 12 sets of samples (P < 0.05). (B) Gap junction activity. SL/DT assay. After N/N1003A cells were treated with or without 25 ng/mL IGF-I for 0, 10, 20, and 30 minutes, the cells were used in SL/DT assays to evaluate gap junction activity. Top: sample images from each time point. Bottom: gap junction activity was decreased by 44.6% in 30 minutes. Data are the mean ± SD of results in duplicate experiments. Results were statistically significant in eight sets of samples (P < 0.05). (C) Cx43 levels in IGF-I activation cells. N/N1003A cells were harvested with or without treatment of 25 ng/mL of IGF-I for 0, 10, 20, and 30 minutes. In each well, 20 μg protein of the whole-cell lysates was loaded. Proteins were separated by 7.5% SDS-PAGE. Western blot analysis was performed with anti-Cx43 antibody used as the probe. Total cell Cx43 was visualized. No decrease of Cx43 occurred in IGF-I-treated cells. Scale bar: (A) 15 μm; (B) 30 μm.
Figure 4.
 
IGF-I decreased cell surface gap junction plaques and gap junction activity. (A) IGF-I (25 ng/mL) decreased the number of cell surface Cx43 plaques per square micrometer of the measured area. N/N1003A cells were treated with or without 25 ng/mL of IGF-I for 0, 10, 20, and 30 minutes, and cells were then fixed with 2.5% paraformaldehyde in PBS for 10 minutes, as described previously. 9 Top: sample images from each time point. Bottom: after incubation for 30 minutes with IGF-I, cell surface Cx43 plaques were reduced by 57.2%. Data are the mean ± SD of results in triplicate experiments. The results were statistically significant in 12 sets of samples (P < 0.05). (B) Gap junction activity. SL/DT assay. After N/N1003A cells were treated with or without 25 ng/mL IGF-I for 0, 10, 20, and 30 minutes, the cells were used in SL/DT assays to evaluate gap junction activity. Top: sample images from each time point. Bottom: gap junction activity was decreased by 44.6% in 30 minutes. Data are the mean ± SD of results in duplicate experiments. Results were statistically significant in eight sets of samples (P < 0.05). (C) Cx43 levels in IGF-I activation cells. N/N1003A cells were harvested with or without treatment of 25 ng/mL of IGF-I for 0, 10, 20, and 30 minutes. In each well, 20 μg protein of the whole-cell lysates was loaded. Proteins were separated by 7.5% SDS-PAGE. Western blot analysis was performed with anti-Cx43 antibody used as the probe. Total cell Cx43 was visualized. No decrease of Cx43 occurred in IGF-I-treated cells. Scale bar: (A) 15 μm; (B) 30 μm.
Figure 5.
 
Effects of TMB-8, a calcium mobilization inhibitor, on PKCγ translocation induced by IGF-I. (A) Dose curve and time course of translocation of PKCγ stimulated by CaCl2. The CaCl2 doses (0–25 mM, 25 minutes) and time intervals (0–30 minutes, 25 mM) are indicated. The PKCγ antibody was used as the probe in Western blot analysis. Quantitative analysis of mean results (±SD) from three such experiments are also presented. Protein load per lane, 10 μg. (B) Regulation of PKCγ translocation by TMB-8. The inhibition of calcium mobilization was performed by adding 0, 10, and 35 μM of TMB-8 to the treatments 15 minutes before addition of 10 mM CaCl2, 25 ng/mL IGF-I, or 1 μM DAG. The control cells were just treated with TMB-8 at 10, 35, and 70 μm. The cells were harvested at 30 minutes of incubation, and PKCγ translocation was determined by Western blot with anti-PKCγ as the probe. Calcium mobilization was not essential for PKCγ translocation stimulated by IGF-I or DAG, but it was necessary for PKCγ translocation induced by exogenous CaCl2. Protein load, 10 μg/lane.
Figure 5.
 
Effects of TMB-8, a calcium mobilization inhibitor, on PKCγ translocation induced by IGF-I. (A) Dose curve and time course of translocation of PKCγ stimulated by CaCl2. The CaCl2 doses (0–25 mM, 25 minutes) and time intervals (0–30 minutes, 25 mM) are indicated. The PKCγ antibody was used as the probe in Western blot analysis. Quantitative analysis of mean results (±SD) from three such experiments are also presented. Protein load per lane, 10 μg. (B) Regulation of PKCγ translocation by TMB-8. The inhibition of calcium mobilization was performed by adding 0, 10, and 35 μM of TMB-8 to the treatments 15 minutes before addition of 10 mM CaCl2, 25 ng/mL IGF-I, or 1 μM DAG. The control cells were just treated with TMB-8 at 10, 35, and 70 μm. The cells were harvested at 30 minutes of incubation, and PKCγ translocation was determined by Western blot with anti-PKCγ as the probe. Calcium mobilization was not essential for PKCγ translocation stimulated by IGF-I or DAG, but it was necessary for PKCγ translocation induced by exogenous CaCl2. Protein load, 10 μg/lane.
Figure 6.
 
DAG activation of PKCγ. (A) Exogenous DAG stimulated translocation of both PKCα and PKCγ at different doses. Top: N/N1003A cells were treated with DAG, as indicated at 0 to 2.5 μM for 30 minutes, and the samples were subjected to translocation studies and probed with antibodies against PKCγ and -α, respectively. Bottom: time course of PKCγ translocation by 1 μM DAG. Quantitative analyses of results in three experiments (±SD) are shown beneath each gel. (B) IGF-I (25 ng/mL) increased internal DAG levels early. N/N1003A cells were grown in the absence or presence of 25 ng/mL of IGF-I added at 5, 10, 20, 30 minutes. The whole-cell lipids were extracted with chloroform-methanol and used as the substrate for DAG kinase. The product, 32P-phosphatidic acid, was quantitated, and cellular DAG levels were calculated. Mean results in triplicate experiments were statistically significant (P < 0.05).
Figure 6.
 
DAG activation of PKCγ. (A) Exogenous DAG stimulated translocation of both PKCα and PKCγ at different doses. Top: N/N1003A cells were treated with DAG, as indicated at 0 to 2.5 μM for 30 minutes, and the samples were subjected to translocation studies and probed with antibodies against PKCγ and -α, respectively. Bottom: time course of PKCγ translocation by 1 μM DAG. Quantitative analyses of results in three experiments (±SD) are shown beneath each gel. (B) IGF-I (25 ng/mL) increased internal DAG levels early. N/N1003A cells were grown in the absence or presence of 25 ng/mL of IGF-I added at 5, 10, 20, 30 minutes. The whole-cell lipids were extracted with chloroform-methanol and used as the substrate for DAG kinase. The product, 32P-phosphatidic acid, was quantitated, and cellular DAG levels were calculated. Mean results in triplicate experiments were statistically significant (P < 0.05).
Figure 7.
 
DAG decreased cell surface gap junction plaques and gap junction activity. (A) DAG (1 μM) decreased the number of cell surface Cx43 plaques per square micrometer of the measured area. N/N1003A cells were treated with or without 1 μM DAG for 0, 15, and 30 minutes, and the cells were then fixed with 2.5% paraformaldehyde in PBS for 10 minutes as described previously. 12 Top: sample images. Bottom: after incubation with DAG, the cell surface Cx43 plaques were reduced by 40.6% within 30 minutes. The mean ± SD of results in six experiments was statistically significant (P < 0.05). (B) SL/DT assay. After N/N1003A cells were treated with or without 1 μM DAG for 0, 15, and 30 minutes, the cells were used for SL/DT assays to evaluate the gap junction activity. Top: sample images. Bottom: gap junction activity decreased 42.9% in DAG-treated cells for 30 minutes. The mean ± SD of results in six experiments were statistically significant (P < 0.05). Scale bar: (A) 15 μm; (B) 30 μm.
Figure 7.
 
DAG decreased cell surface gap junction plaques and gap junction activity. (A) DAG (1 μM) decreased the number of cell surface Cx43 plaques per square micrometer of the measured area. N/N1003A cells were treated with or without 1 μM DAG for 0, 15, and 30 minutes, and the cells were then fixed with 2.5% paraformaldehyde in PBS for 10 minutes as described previously. 12 Top: sample images. Bottom: after incubation with DAG, the cell surface Cx43 plaques were reduced by 40.6% within 30 minutes. The mean ± SD of results in six experiments was statistically significant (P < 0.05). (B) SL/DT assay. After N/N1003A cells were treated with or without 1 μM DAG for 0, 15, and 30 minutes, the cells were used for SL/DT assays to evaluate the gap junction activity. Top: sample images. Bottom: gap junction activity decreased 42.9% in DAG-treated cells for 30 minutes. The mean ± SD of results in six experiments were statistically significant (P < 0.05). Scale bar: (A) 15 μm; (B) 30 μm.
The authors thank John Redden (Oakland University, Rochester, MI) for the gift of N/N1003A rabbit lens epithelial cell line. 
Wagner, L, Saleh, SM, Boyle, DL, Takemoto, DJ. (2002) Effect of protein kinase Cγ on gap junction disassembly in lens epithelial cells and retinal cells in culture Mol Vis 8,59-66 [PubMed]
Simon, AM, Goodenough, DA. (1998) Diverse function of vertebrate gap junctions Trends Cell Biol 8,477-483 [CrossRef] [PubMed]
Donaldson, P, Eckert, R, Green, C, Kistler, J. (1997) Gap junction channels: new roles in disease Histol Histopathol 12,219-231 [PubMed]
Gong, X, Agopian, K, Kumar, NM, Gilula, NB. (1999) Genetic factors influence cataract formation in α3 connexin knockout mice Dev Genet 24,27-32 [CrossRef] [PubMed]
Gong, X, Baldo, GJ, Kumar, NM, Gilula, NB, Mathias, RT. (1998) Gap junctional coupling in lenses lacking α3 connexin Proc Natl Acad Sci USA 95,15303-15308 [CrossRef] [PubMed]
Baruch, A, Greenbaum, D, Levy, ET, et al (2001) Defining a link between gap junction communication, proteolysis, and cataract formation J Biol Chem 276,28999-29006 [CrossRef] [PubMed]
Kistler, J, Lin, JS, Bond, J, et al (1999) Connexins in the lens: are they to blame in diabetic cataractogenesis? Novartis Found Symp 219,97-108 [PubMed]
Gonzalez, K, Udovichenko, I, Cunnick, J, Takemoto, DJ. (1993) Protein kinase C in galactosemic and tolrestat-treated lens epithelial cells Curr Eye Res 12,373-377 [CrossRef] [PubMed]
Saleh, SM, Takemoto, DJ. (2000) Overexpression of protein kinase Cγ inhibits gap junctional intercellular communication in the lens epithelial cells Exp Eye Res 77,99-102
Lopaczynski, W. (1999) Differential regulation of signaling pathways for insulin and insulin-like growth factor I Acta Biochim Pol 46,51-60 [PubMed]
Hopfner, M, Berger, A, Folsch, UR, Loser, C. (2002) Effects of insulin-like growth factor I on growth and polyamine metabolism in various organs in rats Digestion 65,103-111 [CrossRef] [PubMed]
Hadsell, DL, Abdel-Fattah, G. (2001) Regulation of cell apoptosis by insulin-like growth factor I Adv Exp Med Biol 501,79-85 [PubMed]
Jones, JI, Clemmons, DR. (1995) Insulin-like growth factors and their binding proteins: biological actions Endocr Rev 16,3-34 [PubMed]
Kojima, I, Matsunaga, H, Kurokawa, K, Ogata, E, Nishimoto, I. (1988) Calcium influx: an intracellular message of the mitogenic action of insulin-like growth factor-I J Biol Chem 263,16561-16567 [PubMed]
Kojima, I, Kitaoka, M, Ogata, E. (1990) Insulin-like growth factor-I stimulates diacylglycerol production via multiple pathways in Balb/c 3T3 cells J Biol Chem 265,16846-16850 [PubMed]
Kojima, I, Mogami, H, Shibata, H, Ogata, E. (1993) Role of calcium entry and protein kinase C in the progression activity of insulin-like growth factor-I in Balb/c 3T3 cells J Biol Chem 268,10003-10006 [PubMed]
Neri, LM, Borgatti, P, Capitani, S, Martelli, AM. (1998) Nuclear diacylglycerol produced by phosphoinositide-specific phospholipase C is responsible for nuclear translocation of protein kinase C-α J Biol Chem 273,29768-29744
Grigorescu, F, Baccara, MT, Rouard, M, Renard, E. (1994) Insulin and IGF-I signaling in oocyte maturation Horm Res 42,55-61 [CrossRef] [PubMed]
Warn-Cramer, B, Lampe, P, Kurata, W, et al (1996) Characterization of the mitogen-activated protein kinase phosphorylation sites on the connexin 43 gap junction protein J Biol Chem 271,3779-3786 [CrossRef] [PubMed]
Rae, JL, Bartling, C, Rae, J, Mathias, RT. (1996) Dye transfer between cells of lens J Membr Biol 150,89-103 [CrossRef] [PubMed]
Musil, LS, Beyer, EC, Goodenough, DA. (1990) Expression of the gap junction protein connexin 43 in embryonic chicks lens: molecular cloning, ultrastructural localization, and post-translational phosphorylation J Membr Biol 116,163-175 [CrossRef] [PubMed]
Takens-Kwak, B, Jongsma, H. (1992) Cardiac gap junctions: three distinct signal channel conductances and their modulation by phosphorylating treatments Pflugers Arch 422,198-200 [CrossRef] [PubMed]
Moreno, A, Fishman, G, Spray, D. (1992) Phosphorylation shifts unitary conductance and modifies voltage dependent kinetics of human connexin 43 gap junction channels Biophys J 62,51-53 [CrossRef] [PubMed]
TenBroek, EM, Louis, CF, Johnson, R. (1997) The differential effects of 12-O-tetradecanoylphorbol-13-acetate on the gap junctions and connexins of the developing mammalian lens Dev Biol 191,88-102 [CrossRef] [PubMed]
Slater, SJ, Ho, C, Kelly, MB, et al (1996) Protein kinase C α contains two activator binding sites that bind phorbol esters and diacylglycerols with opposite affinities J Biol Chem 271,4626-4631
Slater, SJ, Milano, SK, Stagliano, BA, et al (1999) Synergistic activation of protein kinase C-α, -βI, and -γ isoforms induced by diacylglycerol and phorbol ester: roles of membrane association and activating conformational changes Biochem 38,3804-3815 [CrossRef]
Inoguchi, T, Ueda, F, Umeda, F, Yamashita, T, Nawata, H. (1995) Inhibition of intercellular communication via gap junctions in cultured aortic endothelial cells by elevated glucose and phorbol esters Biochem Biophys Res Commun 208,492-497 [CrossRef] [PubMed]
Xia, P, Inoguchi, T, Kern, T, Engerman, R, Oates, P, King, G. (1994) Characterization of the mechanism for the chronic activation of diacylglycerol-protein kinase C pathway in diabetes and hypergalactosemia Diabetes 43,1122-1129 [CrossRef] [PubMed]
Kuriko, T, Inoguchi, T, Umeda, F, Ueda, F, Nawata, H. (1998) High glucose induces alternations of gap junction permeability and phosphorylation of connexin 43 in cultured aortic smooth muscle cells Diabetes 47,931-936 [CrossRef] [PubMed]
Klok, EJ, Lubsen, NH, Chamberlain, CG, McAvoy, JW. (1998) Induction and maintenance of differentiation of rat lens epithelium by FGF-2, insulin and IGF-I Exp Eye Res 67,425-431 [CrossRef] [PubMed]
Claeys, I, Simonet, G, Poels, J, et al (2002) Insulin-related peptides and their conserved signal transduction pathway Peptides 23,807-816 [CrossRef] [PubMed]
Feldman, EL, Sullivan, KA, Kim, B, Russell, JW. (1997) Insulin-like growth factors regulate neuronal differentiation and survival Neurobiol Dis 4,201-214 [CrossRef] [PubMed]
Teruel, MN, Meyer, T. (2002) Parallel single-cell monitoring of receptor-triggered membrane translocation of a calcium-sensing protein module Science 295,1910-1912 [CrossRef] [PubMed]
Cho, W. (2001) Membrane targeting by C1 and C2 domains J Biol Chem 276,32407-32410 [CrossRef] [PubMed]
Oancea, E, Meyer, T. (1998) Protein kinase C as a molecular machine for decoding calcium and diacylglycerol signals Cell 95,307-318 [CrossRef] [PubMed]
Hunn, M, Quest, A. (1997) Cysteine-rich regions of protein kinase C delta are functionally non-equivalent: differences between cysteine-rich regions of non-calcium dependent protein kinase C delta and calcium-dependent protein kinase C gamma FEBS Lett 400,226-232 [CrossRef] [PubMed]
Homma, N, Alvarado, JL, Coombs, W, et al (1998) A particle-receptor model for the insulin-induced closure of connexin43 channels Circ Res 83,27-32 [CrossRef] [PubMed]
Warn-Cramer, BJ, Cottrel, GT, Burt, JM, Lau, AF. (1998) Regulation of connexin 43 gap junctional communication by mitogen-activated protein kinase J Biol Chem 273,9188-9196 [CrossRef] [PubMed]
Zhou, L, Kasperek, EM, Nicholson, BJ. (1999) Dissection of the molecular basis of pp60 (v-src) induced gating of connexin 43 gap junction channels J Cell Biol 144,1033-1045 [CrossRef] [PubMed]
Figure 1.
 
IGF-I stimulated translocation of PKCγ, but not of PKCα. (A) N/N1003A cells were treated with 0, 1, 5, 10, 25, and 50 ng/mL IGF-I after 20 minutes of incubation. Proteins (10 μg) of membrane or supernatant fractions were separated on 7.5% SDS-PAGE and Western blot analysis performed with antibodies against PKCα or -γ. IGF-I induced translocation of PKCγ, but not of PKCα, to the membrane fractions. Cells were treated with acidic FGF (aFGF) as a negative control to demonstrate no translocation of PKCγ or -α. (B) The N/N1003A cells were treated with or without 25 ng/mL IGF-I for 0, 5, 10, 20, or 30 minutes. The supernatant and membrane fractions were prepared and Western blots were probed with PKCγ antibody. The cells were collected at each time interval, separated into supernatants and membrane fractions, and subjected to Western blot analysis to detect the translocation of PKCγ. Time courses of PKCγ translocation are shown. (C) Quantitative analysis of results in three experiments (±SD), showing PKCγ translocation in a dose- and time- dependent manner.
Figure 1.
 
IGF-I stimulated translocation of PKCγ, but not of PKCα. (A) N/N1003A cells were treated with 0, 1, 5, 10, 25, and 50 ng/mL IGF-I after 20 minutes of incubation. Proteins (10 μg) of membrane or supernatant fractions were separated on 7.5% SDS-PAGE and Western blot analysis performed with antibodies against PKCα or -γ. IGF-I induced translocation of PKCγ, but not of PKCα, to the membrane fractions. Cells were treated with acidic FGF (aFGF) as a negative control to demonstrate no translocation of PKCγ or -α. (B) The N/N1003A cells were treated with or without 25 ng/mL IGF-I for 0, 5, 10, 20, or 30 minutes. The supernatant and membrane fractions were prepared and Western blots were probed with PKCγ antibody. The cells were collected at each time interval, separated into supernatants and membrane fractions, and subjected to Western blot analysis to detect the translocation of PKCγ. Time courses of PKCγ translocation are shown. (C) Quantitative analysis of results in three experiments (±SD), showing PKCγ translocation in a dose- and time- dependent manner.
Figure 2.
 
Interaction between PKCγ and Cx43 was enhanced by IGF-I. (A) N/N1003A cells were harvested with or without treatment of 25 ng/mL of IGF-I for 30 minutes. The whole-cell lysates (3 mg protein) were incubated with 5 μg PKCγ antibody or Cx43 antibody at 4°C overnight. Top: Coimmunoprecipitation complexes were subjected to Western blot with anti-Cx43 or anti-PKCγ used as probes. Bottom: Quantitative analysis of results in three experiments (±SD), showing IGF-I-enhanced coimmunoprecipitation of PKCγ and Cx43. IP, immunoprecipitation; IB, immunoblot. (B) Negative controls for coimmunoprecipitation. Lane 1: anti-PKCγ antisera+whole-cell extracts (3 mg protein)+protein-agarose beads; lane 2: whole-cell extracts+protein-agarose beads; lane 3: protein-agarose beads; lane 4: nonimmunoprecipitated whole-cell extracts (10 μg); lane 5: anti-PKCγ antisera+proteinagarose beads. For Western blot, anti-PKCγ was used as a primary antibody and anti-mouse IgG as a secondary antibody, and results were visualized with chemiluminescence. (C) The negative control for coimmunoprecipitation. Lane 1: anti-Cx43 antisera+whole-cell extracts (3 mg protein)+protein-agarose beads; lane 2: nonimmunoprecipitated whole-cell extracts (10 μg); lane 3: whole-cell extracts+protein-agarose beads; lane 4: protein-agarose beads; lane 5: anti-Cx43 antisera+protein-agarose beads. For Western blot, anti-Cx43 was used as a primary antibody and anti-rabbit IgG as a secondary antibody, with results visualized with chemiluminescence.
Figure 2.
 
Interaction between PKCγ and Cx43 was enhanced by IGF-I. (A) N/N1003A cells were harvested with or without treatment of 25 ng/mL of IGF-I for 30 minutes. The whole-cell lysates (3 mg protein) were incubated with 5 μg PKCγ antibody or Cx43 antibody at 4°C overnight. Top: Coimmunoprecipitation complexes were subjected to Western blot with anti-Cx43 or anti-PKCγ used as probes. Bottom: Quantitative analysis of results in three experiments (±SD), showing IGF-I-enhanced coimmunoprecipitation of PKCγ and Cx43. IP, immunoprecipitation; IB, immunoblot. (B) Negative controls for coimmunoprecipitation. Lane 1: anti-PKCγ antisera+whole-cell extracts (3 mg protein)+protein-agarose beads; lane 2: whole-cell extracts+protein-agarose beads; lane 3: protein-agarose beads; lane 4: nonimmunoprecipitated whole-cell extracts (10 μg); lane 5: anti-PKCγ antisera+proteinagarose beads. For Western blot, anti-PKCγ was used as a primary antibody and anti-mouse IgG as a secondary antibody, and results were visualized with chemiluminescence. (C) The negative control for coimmunoprecipitation. Lane 1: anti-Cx43 antisera+whole-cell extracts (3 mg protein)+protein-agarose beads; lane 2: nonimmunoprecipitated whole-cell extracts (10 μg); lane 3: whole-cell extracts+protein-agarose beads; lane 4: protein-agarose beads; lane 5: anti-Cx43 antisera+protein-agarose beads. For Western blot, anti-Cx43 was used as a primary antibody and anti-rabbit IgG as a secondary antibody, with results visualized with chemiluminescence.
Figure 3.
 
Cx43 phosphorylation was enhanced by IGF-I. (A) In vitro phosphorylation of Cx43. N/N1003A cells were treated with 25 ng/mL IGF-I and harvested at the time intervals shown. Primary antibodies (5 μg) were added to 1 mL of whole-cell extracts containing 3 μg/μL total proteins for immunoprecipitation. Top: the protein complexes were used for in vitro phosphorylation. Bottom: the results of three experiments ± SD were subjected to quantitative analysis. IGF-I caused enhanced PKCγ activity that resulted in phosphorylation of Cx43. The phosphorylation levels reached maximum by 20 minutes after IGF-I treatment. p-Cx43, 32P-labeled Cx43. (B) In vivo phosphorylation of Cx43. Top: whole-cell Cx43 was immunoprecipitated from samples of N/N1003A cells treated with 25 ng/mL of IGF-I at 0, 10, 20, and 30 minutes. The immunoprecipitated Cx43 was analyzed by 12.5% SDS-PAGE and Western blot, probed with anti-phosphoserine antisera. The cells treated with IGF-I had an increase in serine phosphorylation of Cx43 compared with control cells after 30 minutes. Bottom: mean ± SD of results obtained in three experiments. (C) Top: the blot was then stripped and reprobed with anti-Cx43 to show the equal loading for Cx43. Bottom: mean ± SD of results obtained in three experiments. IP, immunoprecipitation; IB, immunoblot.
Figure 3.
 
Cx43 phosphorylation was enhanced by IGF-I. (A) In vitro phosphorylation of Cx43. N/N1003A cells were treated with 25 ng/mL IGF-I and harvested at the time intervals shown. Primary antibodies (5 μg) were added to 1 mL of whole-cell extracts containing 3 μg/μL total proteins for immunoprecipitation. Top: the protein complexes were used for in vitro phosphorylation. Bottom: the results of three experiments ± SD were subjected to quantitative analysis. IGF-I caused enhanced PKCγ activity that resulted in phosphorylation of Cx43. The phosphorylation levels reached maximum by 20 minutes after IGF-I treatment. p-Cx43, 32P-labeled Cx43. (B) In vivo phosphorylation of Cx43. Top: whole-cell Cx43 was immunoprecipitated from samples of N/N1003A cells treated with 25 ng/mL of IGF-I at 0, 10, 20, and 30 minutes. The immunoprecipitated Cx43 was analyzed by 12.5% SDS-PAGE and Western blot, probed with anti-phosphoserine antisera. The cells treated with IGF-I had an increase in serine phosphorylation of Cx43 compared with control cells after 30 minutes. Bottom: mean ± SD of results obtained in three experiments. (C) Top: the blot was then stripped and reprobed with anti-Cx43 to show the equal loading for Cx43. Bottom: mean ± SD of results obtained in three experiments. IP, immunoprecipitation; IB, immunoblot.
Figure 4.
 
IGF-I decreased cell surface gap junction plaques and gap junction activity. (A) IGF-I (25 ng/mL) decreased the number of cell surface Cx43 plaques per square micrometer of the measured area. N/N1003A cells were treated with or without 25 ng/mL of IGF-I for 0, 10, 20, and 30 minutes, and cells were then fixed with 2.5% paraformaldehyde in PBS for 10 minutes, as described previously. 9 Top: sample images from each time point. Bottom: after incubation for 30 minutes with IGF-I, cell surface Cx43 plaques were reduced by 57.2%. Data are the mean ± SD of results in triplicate experiments. The results were statistically significant in 12 sets of samples (P < 0.05). (B) Gap junction activity. SL/DT assay. After N/N1003A cells were treated with or without 25 ng/mL IGF-I for 0, 10, 20, and 30 minutes, the cells were used in SL/DT assays to evaluate gap junction activity. Top: sample images from each time point. Bottom: gap junction activity was decreased by 44.6% in 30 minutes. Data are the mean ± SD of results in duplicate experiments. Results were statistically significant in eight sets of samples (P < 0.05). (C) Cx43 levels in IGF-I activation cells. N/N1003A cells were harvested with or without treatment of 25 ng/mL of IGF-I for 0, 10, 20, and 30 minutes. In each well, 20 μg protein of the whole-cell lysates was loaded. Proteins were separated by 7.5% SDS-PAGE. Western blot analysis was performed with anti-Cx43 antibody used as the probe. Total cell Cx43 was visualized. No decrease of Cx43 occurred in IGF-I-treated cells. Scale bar: (A) 15 μm; (B) 30 μm.
Figure 4.
 
IGF-I decreased cell surface gap junction plaques and gap junction activity. (A) IGF-I (25 ng/mL) decreased the number of cell surface Cx43 plaques per square micrometer of the measured area. N/N1003A cells were treated with or without 25 ng/mL of IGF-I for 0, 10, 20, and 30 minutes, and cells were then fixed with 2.5% paraformaldehyde in PBS for 10 minutes, as described previously. 9 Top: sample images from each time point. Bottom: after incubation for 30 minutes with IGF-I, cell surface Cx43 plaques were reduced by 57.2%. Data are the mean ± SD of results in triplicate experiments. The results were statistically significant in 12 sets of samples (P < 0.05). (B) Gap junction activity. SL/DT assay. After N/N1003A cells were treated with or without 25 ng/mL IGF-I for 0, 10, 20, and 30 minutes, the cells were used in SL/DT assays to evaluate gap junction activity. Top: sample images from each time point. Bottom: gap junction activity was decreased by 44.6% in 30 minutes. Data are the mean ± SD of results in duplicate experiments. Results were statistically significant in eight sets of samples (P < 0.05). (C) Cx43 levels in IGF-I activation cells. N/N1003A cells were harvested with or without treatment of 25 ng/mL of IGF-I for 0, 10, 20, and 30 minutes. In each well, 20 μg protein of the whole-cell lysates was loaded. Proteins were separated by 7.5% SDS-PAGE. Western blot analysis was performed with anti-Cx43 antibody used as the probe. Total cell Cx43 was visualized. No decrease of Cx43 occurred in IGF-I-treated cells. Scale bar: (A) 15 μm; (B) 30 μm.
Figure 5.
 
Effects of TMB-8, a calcium mobilization inhibitor, on PKCγ translocation induced by IGF-I. (A) Dose curve and time course of translocation of PKCγ stimulated by CaCl2. The CaCl2 doses (0–25 mM, 25 minutes) and time intervals (0–30 minutes, 25 mM) are indicated. The PKCγ antibody was used as the probe in Western blot analysis. Quantitative analysis of mean results (±SD) from three such experiments are also presented. Protein load per lane, 10 μg. (B) Regulation of PKCγ translocation by TMB-8. The inhibition of calcium mobilization was performed by adding 0, 10, and 35 μM of TMB-8 to the treatments 15 minutes before addition of 10 mM CaCl2, 25 ng/mL IGF-I, or 1 μM DAG. The control cells were just treated with TMB-8 at 10, 35, and 70 μm. The cells were harvested at 30 minutes of incubation, and PKCγ translocation was determined by Western blot with anti-PKCγ as the probe. Calcium mobilization was not essential for PKCγ translocation stimulated by IGF-I or DAG, but it was necessary for PKCγ translocation induced by exogenous CaCl2. Protein load, 10 μg/lane.
Figure 5.
 
Effects of TMB-8, a calcium mobilization inhibitor, on PKCγ translocation induced by IGF-I. (A) Dose curve and time course of translocation of PKCγ stimulated by CaCl2. The CaCl2 doses (0–25 mM, 25 minutes) and time intervals (0–30 minutes, 25 mM) are indicated. The PKCγ antibody was used as the probe in Western blot analysis. Quantitative analysis of mean results (±SD) from three such experiments are also presented. Protein load per lane, 10 μg. (B) Regulation of PKCγ translocation by TMB-8. The inhibition of calcium mobilization was performed by adding 0, 10, and 35 μM of TMB-8 to the treatments 15 minutes before addition of 10 mM CaCl2, 25 ng/mL IGF-I, or 1 μM DAG. The control cells were just treated with TMB-8 at 10, 35, and 70 μm. The cells were harvested at 30 minutes of incubation, and PKCγ translocation was determined by Western blot with anti-PKCγ as the probe. Calcium mobilization was not essential for PKCγ translocation stimulated by IGF-I or DAG, but it was necessary for PKCγ translocation induced by exogenous CaCl2. Protein load, 10 μg/lane.
Figure 6.
 
DAG activation of PKCγ. (A) Exogenous DAG stimulated translocation of both PKCα and PKCγ at different doses. Top: N/N1003A cells were treated with DAG, as indicated at 0 to 2.5 μM for 30 minutes, and the samples were subjected to translocation studies and probed with antibodies against PKCγ and -α, respectively. Bottom: time course of PKCγ translocation by 1 μM DAG. Quantitative analyses of results in three experiments (±SD) are shown beneath each gel. (B) IGF-I (25 ng/mL) increased internal DAG levels early. N/N1003A cells were grown in the absence or presence of 25 ng/mL of IGF-I added at 5, 10, 20, 30 minutes. The whole-cell lipids were extracted with chloroform-methanol and used as the substrate for DAG kinase. The product, 32P-phosphatidic acid, was quantitated, and cellular DAG levels were calculated. Mean results in triplicate experiments were statistically significant (P < 0.05).
Figure 6.
 
DAG activation of PKCγ. (A) Exogenous DAG stimulated translocation of both PKCα and PKCγ at different doses. Top: N/N1003A cells were treated with DAG, as indicated at 0 to 2.5 μM for 30 minutes, and the samples were subjected to translocation studies and probed with antibodies against PKCγ and -α, respectively. Bottom: time course of PKCγ translocation by 1 μM DAG. Quantitative analyses of results in three experiments (±SD) are shown beneath each gel. (B) IGF-I (25 ng/mL) increased internal DAG levels early. N/N1003A cells were grown in the absence or presence of 25 ng/mL of IGF-I added at 5, 10, 20, 30 minutes. The whole-cell lipids were extracted with chloroform-methanol and used as the substrate for DAG kinase. The product, 32P-phosphatidic acid, was quantitated, and cellular DAG levels were calculated. Mean results in triplicate experiments were statistically significant (P < 0.05).
Figure 7.
 
DAG decreased cell surface gap junction plaques and gap junction activity. (A) DAG (1 μM) decreased the number of cell surface Cx43 plaques per square micrometer of the measured area. N/N1003A cells were treated with or without 1 μM DAG for 0, 15, and 30 minutes, and the cells were then fixed with 2.5% paraformaldehyde in PBS for 10 minutes as described previously. 12 Top: sample images. Bottom: after incubation with DAG, the cell surface Cx43 plaques were reduced by 40.6% within 30 minutes. The mean ± SD of results in six experiments was statistically significant (P < 0.05). (B) SL/DT assay. After N/N1003A cells were treated with or without 1 μM DAG for 0, 15, and 30 minutes, the cells were used for SL/DT assays to evaluate the gap junction activity. Top: sample images. Bottom: gap junction activity decreased 42.9% in DAG-treated cells for 30 minutes. The mean ± SD of results in six experiments were statistically significant (P < 0.05). Scale bar: (A) 15 μm; (B) 30 μm.
Figure 7.
 
DAG decreased cell surface gap junction plaques and gap junction activity. (A) DAG (1 μM) decreased the number of cell surface Cx43 plaques per square micrometer of the measured area. N/N1003A cells were treated with or without 1 μM DAG for 0, 15, and 30 minutes, and the cells were then fixed with 2.5% paraformaldehyde in PBS for 10 minutes as described previously. 12 Top: sample images. Bottom: after incubation with DAG, the cell surface Cx43 plaques were reduced by 40.6% within 30 minutes. The mean ± SD of results in six experiments was statistically significant (P < 0.05). (B) SL/DT assay. After N/N1003A cells were treated with or without 1 μM DAG for 0, 15, and 30 minutes, the cells were used for SL/DT assays to evaluate the gap junction activity. Top: sample images. Bottom: gap junction activity decreased 42.9% in DAG-treated cells for 30 minutes. The mean ± SD of results in six experiments were statistically significant (P < 0.05). Scale bar: (A) 15 μm; (B) 30 μm.
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