IOVS
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Islam, M.
Right arrow Articles by Akhtar, R. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Islam, M.
Right arrow Articles by Akhtar, R. A.
(Investigative Ophthalmology and Visual Science. 2001;42:1472-1478.)
© 2001 by The Association for Research in Vision and Ophthalmology, Inc.

Upregulation of Phospholipase C{gamma}1 Activity during EGF-Induced Proliferation of Corneal Epithelial Cells: Effect of Phosphoinositide-3 Kinase

Mozaffarul Islam and Rashid A. Akhtar

From the Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
PURPOSE. Previously, the authors showed that epidermal growth factor (EGF) stimulates phospholipase C{gamma}1 (PLC{gamma}1) and phosphoinositide-3 kinase (PI3K) activities in confluent rabbit corneal epithelial cells (RCECs). The purpose of this study was to investigate whether PLC{gamma}1 activity is upregulated during EGF-induced proliferation of RCECs and to determine whether there is any cross-talk between PLC{gamma}1 and PI3K in these cells.

METHODS. Simian virus (SV)-40–immortalized RCECs were cultured in the presence and absence of EGF and other agents. At prescribed time intervals, the cultures were terminated and the cells counted. PLC{gamma}1 activity in intact cells was assessed by measuring the production of [3H]IP3 in [3H]myoinositol-labeled cells. The in vitro enzyme activity was assayed using immunoprecipitated PLC{gamma}1 and [3H]PI(4,5)P2 as substrate. [3H]IP3, the product of PLC{gamma}1, was analyzed by anion-exchange chromatography. The changes in protein content and level of phosphorylation of PLC{gamma}1 were determined by Western immunoblot analysis, with the appropriate antibodies.

RESULTS. Addition of EGF (50 ng/ml) caused a time-dependent increase in proliferation of RCECs. The effect of EGF peaked at approximately 36 hours. Under the same experimental conditions, EGF stimulated PLC{gamma}1 activity with a time course similar to that of cell proliferation. Data from Western immunoblot analysis revealed that the EGF-stimulated PLC{gamma}1 activity was due to increased synthesis of the enzyme. Furthermore, during cell proliferation, tyrosine phosphorylation of PLC{gamma}1 increased in a time-dependent manner that corresponded closely with the expression of PLC{gamma}1. EGF exerted its effects both on cell proliferation and PLC{gamma}1 activation in a dose-dependent manner. Treatment of the cells with U-73122, a PLC inhibitor, or myr-GLYRKAMRLRY, a myristoylated PLC{gamma}1 inhibitor peptide, caused attenuation of both the EGF-stimulated cell proliferation and PLC{gamma}1 activity. Treatment of the cells with the PI3K inhibitors, wortmannin or LY294002, caused inhibition of both EGF-stimulated cell proliferation and PLC{gamma}1 activation. Addition of PI(3,4,5)P3 to the in vitro PLC{gamma}1 assay mixture stimulated the enzyme activity in a dose-dependent manner.

CONCLUSIONS. The data suggest a positive correlation between EGF-stimulated PLC{gamma}1 activation and cell proliferation in RCECs. The EGF-stimulated PLC{gamma}1 activity was mirrored by increased synthesis and tyrosine phosphorylation of the enzyme. The data also show that PLC{gamma}1 activation and cell proliferation were inhibited by PI3K inhibitors, suggesting a role for PI3K in EGF-stimulated proliferation of corneal epithelial cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Stimulation of a variety of cell surface receptors by the appropriate ligand results in activation of phospholipase C (PLC) and phosphoinositide 3-kinase (PI3K) in several cell types.1 2 3 4 PLC catalyzes the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) to generate two intracellular second-messenger molecules, diacylglycerol (DAG) and 1,4,5-trisphosphate (IP3).1 2 IP3 binds to intracellular receptors to initiate calcium release from intracellular stores. DAG, on the other hand, functions as an allosteric activator of protein kinase C (PKC), which is involved in cell proliferation and differentiation. To date, 10 mammalian PLC isozymes have been identified and can be divided into three types: PLCß, PLC{gamma}, and PLC{delta}.2 PLCß subtypes are regulated by heterotrimeric guanosine triphosphate (GTP)-binding proteins of the Gq subfamily. PLC{gamma} isoforms are activated by both receptor and nonreceptor tyrosine kinases, but the regulation of the PLC{delta} family of enzymes is less clear. PI3Ks phosphorylate the hydroxyl group at the 3-position of inositol in phosphoinositides to generate PI(3)P, PI(3,4)P2 and PI(3,4,5)P3.3 4 These phosphoinositides are not hydrolyzed by known PLCs and therefore do not serve to generate additional intracellular second-messenger molecules.

Recently, however, isoforms of phospholipase D, which specifically hydrolyze PI(3,4)P2 and PI(3,4,5)P3 to phosphatidic acid and the corresponding inositol phosphates, have been reported.5 PI(3,4)P2 and PI(3,4,5)P3 are present only in trace amounts in normal resting cells, but their levels increase several fold in response to many types of stimuli.3 Several studies using genetics, PI3K inhibitors, and PI3K overexpression have implicated the PI3Ks in the regulation of cell growth and differentiation, cell survival, cytoskeletal reorganization, and membrane trafficking. PI3Ks have been classified into three types on the basis of their primary structure, regulation and in vitro substrate specificity.4 Type I PI3Ks are heterodimers consisting of p85 regulatory and p110 catalytic subunits. They can phosphorylate PI, PI(4)P, and PI(4,5)P2, and are activated by receptor and nonreceptor tyrosine kinases and G-protein–linked receptors. Type II PI3Ks have substrate specificity restricted to PI and PI(4)P, and their mode of activation remains obscure. Type III PI3Ks are homologues of Saccharomyces cerevisiae Vps34p, which phosphorylates PI exclusively and is activated by a serine/threonine (Ser/Thr) kinase.

It is known that interaction of epidermal growth factor (EGF) with its receptor is followed by receptor dimerization and phosphorylation of its tyrosine residues, which provides docking sites for several SH2-containing proteins, including PLC{gamma}1 and PI3K.2 6 The recruitment of PLC{gamma}1 and PI3K to EGF receptors results in their tyrosine phosphorylation and activation. Regeneration of corneal epithelium after in vivo injury begins with the migration of epithelial cells to the wounded area. This is followed by mitosis and cell proliferation to repair the defect. We and others have shown that topical application of EGF to the injured cornea stimulates cell proliferation and enhances wound repair.7 8 The same outcome results from the addition of EGF to corneal epithelial cells in culture.9 10

The exact biochemical events leading from EGF-receptor interaction and culminating in enhanced cell proliferation are not well understood. Previously, we reported that addition of EGF to quiescent corneal epithelial cells causes activation of PLC{gamma}1 and PI3K.11 12 Recently, we showed that activation of PI3K by EGF, both in vivo and in vitro, correlates positively with cell proliferation and wound repair in rabbit corneal epithelium.8 10 The objective of the present study was to investigate whether there is any correlation between PLC{gamma}1 activation and corneal epithelial cell proliferation induced by EGF. In addition, we examined the effects of PI3K on PLC{gamma}1 activation in the EGF-treated epithelial cells. For this work, we used simian virus (SV)-40–immortalized rabbit corneal epithelial cells (RCECs), which are capable of growing for many passages without any alteration in their morphology or biochemical characteristics.13 14


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Dulbecco’s modified Eagle’s medium (DMEM), insulin, and gentamicin were purchased from Gibco (Grand Island, NY); human recombinant EGF, leupeptin, aprotinin, protein A (immobilized on Sepharose CL- 4B), and phenylmethylsulfonyl fluoride (PMSF) from Sigma (St. Louis, MO); PLC{gamma}1 and -ß1 polyclonal antibodies from Santa Cruz Biotechnology (Santa Cruz, CA); PI(4,5)P2, PI(3)P, PI(3,4)P2, and PI(3,4,5)P3 from Biomol (Plymouth Meeting, PA); phosphatidylserine (PS) and phosphatidylethanolamine (PE) from Avanti Polar Lipids (Birmingham, AL); and wortmannin, LY294002, U-73122, and U-73343 from Calbiochem (La Jolla, CA). [3H]PIP2 (specific radioactivity 10 Ci/mmol) was purchased from DuPont NEN (Boston, MA) and [3H]thymidine (specific radioactivity 64 Ci/mmol) from ICN Radiochemicals (Irvine, CA).

Cell Culture
The SV-40–transformed RCECs were thawed and suspended in complete medium (DMEM/F-12 containing 40 µg/ml gentamicin, 5 µg/ml insulin, and 10% fetal bovine serum [FBS]) and cultured in a humidified atmosphere of 95% air-5% CO2. The cultures were maintained by changing the medium every other day until the cells became confluent. To initiate subculture, the confluent cells were washed with Ca2+-Mg2+-free warm phosphate-buffered saline (PBS) and treated with 0.1% trypsin-0.02% EDTA for 5 minutes at 37°C. Next, complete medium was added and the cell suspension centrifuged at 300g for 5 minutes. The pelleted cells were resuspended in complete medium and then seeded in a 25-cm2 culture flask at a density of 2 x 104 cells/cm2. The cultures were maintained by changing the medium on alternate days.

Cell Proliferation Assay
Cell proliferation was assessed by counting (model Z1, Coulter, Hialeah, FL). The cells were washed with warm Ca2+- and Mg2+-free PBS, trypsinized, and suspended in 2 ml complete medium. A portion (100 µl) of the cell suspension was used for cell counting. Triplicate counts were taken for each data point. The incorporation of [3H]thymidine into DNA was measured as described previously.10 Briefly, subconfluent (~60%) cultures were incubated for 36 hours with different concentrations of EGF in DMEM containing 2 µCi/ml [3H]thymidine (specific radioactivity, 2 Ci/mmol). The culture medium was then removed and the cells washed in 10% trichloroacetic acid (TCA). The precipitated DNA was dissolved in 1% SDS-0.3 N NaOH and counted in a scintillation counter.

In Vitro Assay of PLC{gamma}1 and PLCß1
Subconfluent (~60%) cultures were serum starved for 24 hours before treatment with EGF or other agents. At appropriate times, the cultures were terminated and the cells scraped and homogenized in 20 mM Tris-HCl buffer (pH 7.0) containing 5 mM MgCl2, 5 mM EDTA, 1 mM EGTA, 1 mM PMSF, 2 mM Na3VO4, 10 µg/ml leupeptin, and 50 µg/ml aprotinin. The homogenate was centrifuged at 600g for 10 minutes, and the resultant supernatant quantified for protein concentration by the method of Lowry et al.15 Supernatants (cell lysates) containing equal amounts of protein were treated with PLC{gamma}1 or PLCß1 antibody, and the immunoprecipitated protein was used to assay PLC activity as described previously, with minor modifications.16 Briefly, the reaction mixture contained 20 mM Tris-HCl buffer (pH 7.0), 0.1 M NaCl, 2 mM CaCl2, 1 mM EGTA, 1 mM EDTA, 0.1% sodium cholate, 50 µM [3H]PIP2 (30,000 disintegrations per minute [dpm]), and the immunoprecipitated enzyme protein in a final volume of 125 µl. The substrate was prepared by mixing chloroform solutions of [3H]PIP2-unlabeled PIP2, PE, and PS at a molar ratio of 1:2:2, respectively. After evaporation of the solvent under N2, the lipids were suspended by sonication in sodium cholate–containing reaction buffer. The reaction was initiated by addition of the enzyme protein, incubated for 30 minutes at 37°C, and terminated by addition of 0.5 ml chloroform-methanol-concentrated HCl (50:50:1, by volume). Next, 150 µl of 1 M HCl containing 5 mM EGTA was added and the reaction mixture thoroughly mixed and centrifuged. An aliquot (400 µl) of the upper aqueous phase was removed and counted in a scintillation counter. When the effects of PI(3)P, PI(3,4)P2, and PI(3,4,5)P3 on the activity of PLC{gamma}1 or PLCß1 were to be determined, the immunoprecipitated enzymes were treated for 15 minutes with different concentrations of these lipids before their addition to the reaction mixture.

Western Immunoblot Analysis
The protein content and level of tyrosine phosphorylation of PLC{gamma}1 were determined by Western immunoblot analysis, as described previously.11 Briefly, RCECs lysates containing equal amounts of protein from EGF-treated and untreated cells were immunoprecipitated using anti-PLC{gamma}1 antibody. The precipitates were boiled in Laemmli’s buffer for 5 minutes, separated by 10% SDS-PAGE, and the proteins transferred to nitrocellulose membranes. To determine total PLC{gamma}1 content, the membranes were successively blotted with anti-PLC{gamma}1 primary antibody and anti-rabbit horseradish peroxidase (HRP)-conjugated goat secondary antibody. To determine the level of PLC{gamma}1 tyrosine phosphorylation, the blots were stripped and reprobed successively with anti-phosphotyrosine primary antibody and anti-mouse HRP-conjugated secondary antibody. The protein bands were visualized using the enhanced chemiluminescence (ECL) Western detection system (Amersham Pharmacia Biotech, Parsippany, NJ).

Synthesis of Inhibitor Peptides for PLC{gamma}1
The myristoylated PLC{gamma}1 inhibitor peptide, myr-GLYRKMRLRY (myr-PCI(Y)), and its weaker analogue, myr-GLFRKMRLRF (myr-PC I(F)), were synthesized (Pioneer; PerSeptive Biosystems, Framingham, MA) at 0.1 mM, using the 9-fluorenylmethoxycarbonyl (Fmoc) solid phase chemistry. The myristoyl group was added as the free acid. The myristic acid was treated as an amino acid using an extended coupling procedure. Peptide purity was determined by HPLC on a C-18 reversed-phase column and the molecular weight determined by mass spectrometry.

Statistical Analysis of the Data
Each experiment consisted of incubations that, when pooled, yielded three independent samples for each data point. All experiments were performed at least twice, with the results expressed as mean ± SEM. Statistical analysis was performed with Student’s t-test for nonpaired data. P <= 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of EGF on PLC{gamma}1 Activity and Tyrosine Phosphorylation during Cell Proliferation
To determine whether cell counting can provide an accurate measure for cell proliferation, the data from cell counting were compared with data from [3H]thymidine incorporation into DNA. As shown in Table 1 , there was a close correlation between the cell numbers and the [3H]thymidine incorporation into DNA in RCECs treated with different concentrations of EGF. Therefore, in further experiments, we routinely used cell counting to estimate proliferation in RCECs. To investigate whether EGF exerts an effect on PLC{gamma}1 activity during cell proliferation, the serum-starved RCECs were cultured in the presence and absence of EGF, followed by cell counting and determination of PLC{gamma}1 activity. As shown in Figure 1A , in the absence of EGF, there was a gradual increase in cell number that reached a plateau at 36 hours. Addition of EGF (50 ng/ml) exerted a large, time-dependent increase in cell proliferation, compared with the untreated cells. When analyzed for PLC{gamma}1 activity, there was a time-dependent increase in enzyme activity in the untreated cells (Fig. 1B) . Treatment with EGF caused a large increase in PLC{gamma}1 activity, compared with the untreated cells.


View this table:
[in this window]
[in a new window]
  		Table 1. Effect of EGF on Proliferation of RCECs Measured by [3H]Thymidine Incorporation into DNA and by Cell Counting

 


View larger version (24K):
[in this window]
[in a new window]
  		Figure 1. Time-course effect of EGF on PLC{gamma}1 activity during RCEC proliferation. Serum-starved cells were cultured for different time intervals in the absence and presence of EGF (50 ng/ml). (A) Cells were trypsinized and counted. (B) Cells were lysed and the lysate immuno-precipitated with PLC{gamma}1 antibody. The immunoprecipitate was collected and assayed for PLC activity, using [3H]PIP2 as substrate. The enzyme activity is expressed as [3H]IP3 produced per milligram protein in RCECs. (C) Western blot analysis of PLC{gamma}1 immunoprecipitates recovered from cell lysates. (D) Western blot stripped and reprobed with anti-phosphotyrosine antibody. Data are mean ± SEM of three separate experiments with at least duplicate cultures for each data point. *Statistically significant (P < 0.05) increase in cell number or [3H]IP3, compared with the corresponding control.

 
Western blot data revealed a time-dependent increase in PLC{gamma}1 synthesis in the untreated cells (Fig. 1C) . PLC{gamma}1 protein levels were further increased at all time points after treatment with EGF. Reprobing the PLC{gamma}1 immunoblot with phosphotyrosine antibody showed a time-dependent increase in tyrosine phosphorylation of PLC{gamma}1 in EGF-treated and untreated cells (Fig. 1D) . There appeared to be a close correlation between PLC{gamma}1 phosphorylation and the level of PLC{gamma}1 expression. As shown in Figure 2 , EGF caused a dose-dependent increase in cell proliferation and PLC{gamma}1 activity. The effects were observed with as low as 5 ng/ml EGF and were maximal at 50 ng/ml EGF, followed by a slight decrease.



View larger version (21K):
[in this window]
[in a new window]
  		Figure 2. Dose-response effect of EGF on cell proliferation and PLC{gamma}1 activity in RCECs. Serum-starved cells were cultured for 36 hours in the absence and presence of different concentrations of EGF. (A) Cells were trypsinized and counted. (B) Cells were lysed and the lysate immunoprecipitated with PLC{gamma}1 antibody. The immunoprecipitate was collected and assayed for PLC activity using [3H]PIP2 as substrate. Data are mean ± SEM of three independent experiments with triplicate cultures for each data point. *Statistically significant (P < 0.05) increase in cell number, compared with the control.

 
Effect of U-73122 on EGF-Stimulated PLC{gamma}1 Activity and Cell Proliferation
To further examine the relationship between EGF-stimulated PLC{gamma}1 activity and cell proliferation, RCECs were cultured in the presence and absence of EGF and different concentrations of the PLC inhibitor U-73122 and its inactive analogue U-73343. The cultures were terminated and PLC{gamma}1 immunoprecipitated and assayed for enzyme activity in vitro. U-73122 caused a dose-dependent decrease in cell number in EGF-treated RCECs (Fig. 3A) . Maximal inhibition of EGF-stimulated cell proliferation was observed at 2 µM of the inhibitor. When examined for its effect on PLC{gamma}1 activity, U-73122 caused a dose-dependent decrease in PLC{gamma}1 activity in EGF-treated and untreated cells (Fig. 3B) . The stimulatory effect of EGF on PLC{gamma}1 activity was maximally inhibited in the presence of 2 µM U-73122. U-73343, the inactive analogue, failed to exert any significant inhibitory effect on cell proliferation or PLC{gamma}1 activity. U-73122 exerted no effect on EGF-induced expression or tyrosine phosphorylation of PLC{gamma}1 in RCECs during proliferation (Figs. 3C 3D) .



View larger version (30K):
[in this window]
[in a new window]
  		Figure 3. Effect of U-73122 on EGF-stimulated cell proliferation and PLC{gamma}1 activity in RCECs. Serum-starved cells were cultured for 36 hours in the presence and absence of EGF (50 ng/ml) and different concentrations of U-73122. The inactive analogue U-73343 was used as a negative control. (A) Cells were trypsinized and counted. (B) Cells were lysed and immunoprecipitated using PLC{gamma}1 antibody. The resultant immunoprecipitate was assayed for PLC activity using [3H]PIP2 as the substrate. (C) Western blot of the immunoprecipitated PLC{gamma}1. (D) PLC{gamma}1 immunoblot stripped and reprobed with anti-phosphotyrosine antibody. Data are mean ± SEM from three independent experiments with three to four cultures for each data point. *Statistically significant (P < 0.05) decrease in cell number or PLC{gamma}1 activity, compared with the corresponding cultures not treated with U-73122.

 
Effect of Myristoylated PLC Inhibitor Peptide on IP3 Formation and Cell Proliferation
myr-PCI(Y), the myristoylated PLC{gamma}1 inhibitor peptide, was used to further confirm the involvement of PLC{gamma}1 in EGF-stimulated cell proliferation in RCECs. The cells were cultured in the presence of different concentrations of the peptide before counting and determination of PLC{gamma}1 activity in vitro. myr-PCI(Y) inhibited the EGF-induced cell proliferation in a dose-dependent manner (Fig. 4A) . The EGF effect was reduced by approximately 40% in the presence of 10 µM myr-PCI(Y). Unlike U-73122, the peptide did not inhibit the PLC{gamma}1 activity in the untreated cells. However, it inhibited the EGF-induced activation of PLC{gamma}1 in a dose-dependent manner (Fig. 4B) . At 10 µM myr-PCI(Y), the EGF effect was inhibited by almost 50%. myr-PCI(F), a PCI peptide analogue, was considerably less potent in inhibiting the EGF-induced PLC{gamma}1 activation and cell proliferation.



View larger version (30K):
[in this window]
[in a new window]
  		Figure 4. Effect of myristoylated PCI peptides on EGF-stimulated cell proliferation and PLC{gamma}1 activity in RCECs. Serum-starved cells were cultured for 36 hours in the absence and presence of EGF (50 ng/ml) and different concentrations of myr-PCI(Y). myr-PCI(F) was used as a negative control. (A) Cells were trypsinized and counted. (B) Cells were lysed and immunoprecipitated using PLC{gamma}1 antibody. The resultant immunoprecipitate was assayed for PLC activity using [3H]PIP2 as substrate. Data represent mean ± SEM of three independent experiments with at least duplicate cultures for each data point. *Statistically significant (P < 0.05) decrease in cell number or [3H]IP3, compared with the corresponding cultures not treated with myr-PCI(Y).

 
Effect of PI3K Inhibitors on PLC{gamma}1 Activity and Cell Proliferation
We used wortmannin and LY294002 as inhibitors of PI3K to determine whether PLC{gamma}1 lies downstream of PI3K in mediating the effect of EGF on proliferation of corneal epithelial cells. Treatment with wortmannin caused a dose-dependent decrease in EGF-stimulated proliferation of RCECs (Fig. 5A) . Wortmannin also inhibited the EGF-induced activation of PLC{gamma}1in a dose-dependent manner (Fig. 5B) . LY294002 also caused a dose-dependent decrease in EGF-stimulated cell proliferation and PLC{gamma}1 activity in RCECs (Fig. 6) .



View larger version (24K):
[in this window]
[in a new window]
  		Figure 5. Effect of wortmannin on EGF-stimulated cell proliferation and PLC{gamma}1 activity in RCECs. Serum-starved cells were cultured for 36 hours with EGF (50 ng/ml) and different concentrations of wortmannin. (A) Cells were trypsinized and counted. (B) Cells were lysed and immunoprecipitated using PLC{gamma}1 antibody. The resultant immunoprecipitate was assayed for PLC activity using [3H]PIP2 as substrate. Data are mean ± SEM of three to four experiments conducted with at least duplicate cultures for each data point. *Statistically significant (P < 0.05) decrease in cell number or [3H]IP3, compared with the corresponding cultures not treated with wortmannin.

 


View larger version (23K):
[in this window]
[in a new window]
  		Figure 6. Effect of LY294002 on EGF-stimulated cell proliferation and PLC{gamma}1 activity in RCECs. All experimental conditions were the same as described in Figure 5 , except that LY294002 was used as a PI3K inhibitor. Data are mean ± SEM from three to four experiments, each with triplicate cultures. *Statistically significant (P < 0.05) decrease in cell number or [3H]IP3, compared with the corresponding cultures not treated with LY294002.

 
Effect of PI3K Products on the Activity of PLC{gamma}1
We used PI(3)P, PI(3,4)P2, and PI(3,4,5)P3 to investigate whether PLC{gamma}1 could be a direct target of the PI3K products. PLC{gamma}1 was immunoprecipitated from cultured RCECs and assayed for its activity in the presence and absence of 3-phosphoinositides. As shown in Figure 7 , PI(3,4,5)P3 increased the activation of PLC{gamma}1 in a dose-dependent manner up to 100 µM, followed by a slight tapering off. The maximal stimulation, observed at 100 µM, then decreased at higher concentrations of PI(3,4,5)P3. PI(3)P and PI(3,4)P3 did not have any significant stimulatory effect on PLC{gamma}1 activity. Similarly, PI(3,4,5)P3 had no effect on the activity of PLCß1 immunoprecipitated from RCECs (data not shown).



View larger version (19K):
[in this window]
[in a new window]
  		Figure 7. Effects of PI(3)P, PI(3,4)P2, and PI(3,4,5)P3 on PLC{gamma}1 activity. RCECs were lysed and immunoprecipitated with PLC{gamma}1 antibody. The immunoprecipitate was collected and incubated with 3-phosphoinositides for 15 minutes at room temperature. The immunoprecipitate was then assayed for PLC activity using [3H]PIP2 as a substrate. Data are mean ± SEM of three to four experiments, each conducted in triplicate. *Statistically significant (P < 0.05) increase in [3H]IP3, compared with the cultures not treated with 3-phosphoinositides.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Wound repair in corneal epithelium is highly complex and involves cell migration, proliferation and differentiation, and secretion of extracellular matrix proteins. It has been reported that EGF treatment, both in vitro and in vivo, accelerates the wound-healing process by promoting migration and proliferation of the corneal epithelial cells.7 8 The biochemical events that relay signals from the EGF receptor to the nucleus, resulting in increased DNA synthesis, are not clear. It has been reported that addition of EGF to quiescent corneal epithelial cells causes stimulation of PLC{gamma}1, possibly because of its translocation to, and phosphorylation by, the EGF receptor.11

The data from the present study show that serum-starved RCECs can proliferate, albeit at a low rate, in the absence of any exogenously added EGF (Fig. 1A) . The observed proliferative response is most likely due to autocrine secretion of growth-promoting substances by cells in the culture medium. When treated with EGF, the cells exhibited a marked time-dependent increase in cell number. An important finding of the present study is that, under conditions when RCECs were undergoing active proliferation, there was a concomitant increase in PLC{gamma}1 activity both in the EGF-treated and untreated cells (Fig. 1B) . Western blot analysis revealed that the increased PLC{gamma}1 activity was due to increased synthesis and tyrosine phosphorylation of the enzyme (Figs. 1C 1D) . The effect of EGF on PLC was found to be specific for the PLC{gamma}1 isoform. Both the activity and expression of PLCß1 remained unchanged during cell proliferation (data not shown).

These data provide evidence that PLC{gamma}1 is involved in a signaling pathway that mediates the EGF’s effect on DNA synthesis in RCECs. The findings are in accord with previously published reports in which microinjection of anti-PLC{gamma}1 antibody blocked serum- and Ras-stimulated DNA synthesis.17 In other studies, microinjection of PLC{gamma}1 SH2 domains into Madin-Darby canine kidney (MDCK) epithelial cells or fibroblasts blocked the platelet-derived growth factor (PDGF)–induced DNA synthesis.18 19

Additional evidence to support the suggestion that PLC{gamma}1 is involved in corneal epithelial cell proliferation comes from the use of the PLC inhibitor U-73122. This inhibitor has been widely used in a number of studies examining the role of PLC in intracellular signaling mechanisms.20 21 In the present study, by using U-73122, we were able to significantly inhibit the EGF-stimulated PLC{gamma}1 activation, which resulted in a corresponding decrease in EGF-induced proliferation in RCECs (Fig. 3) . The inhibitory effect of U-73122 was not due to the inhibition of PLC{gamma}1 synthesis or its phosphorylation. It has been reported that PLC{gamma}1 possesses, adjacent to its SH2 and SH3 motifs, a PLC inhibitor (PCI) region that strongly suppresses its catalytic activity.22 It has been suggested that stimulation of the cells with growth factors probably dissociates the PCI region of PLC{gamma}1 from the catalytic region, resulting in the activation of the enzyme. Myristoylation of PCI facilitates its entry into Swiss 3T3 cells causing inhibition of cell growth and phosphoinositide hydrolysis.23 We used myr-PCI(Y) to further examine the involvement of PLC{gamma}1 in RCEC proliferation. When added to RCECs, myr-PCI(Y) suppressed the EGF-induced PLC{gamma}1 activation and cell proliferation (Fig. 4) . The inactive structural analogue myr-PCI(F) did not show any consistent inhibitory effect on PLC{gamma}1 activity and cell proliferation, even when used at high concentrations. Taken together, these data provide convincing evidence that PLC{gamma}1 is upregulated during EGF-induced proliferation of RCECs.

Activation of EGF receptor is followed by autophosphorylation of its tyrosine residues, which serve as high-affinity binding sites for SH2-containing proteins, including PLC{gamma}1. Recruitment of PLC{gamma}1 to the receptor results in tyrosine phosphorylation and stimulation of its enzyme activity.2 24 Tyrosine phosphorylation of PLC{gamma}1 has been reported to be critical in transducing mitogenic signals from growth factor receptors to the interior of the cell. In particular, substitution of Phe for Tyr783 completely blocks the activation of PLC{gamma}1 by PDGF in NIH 3T3 cells.25 In addition to being activated by tyrosine phosphorylation, PLC{gamma}1 has been shown to be stimulated by lipid-derived second messengers, such as arachidonic acid, phosphatidic acid, and PI(3,4,5)P3.24 The activity of PLC{gamma}1 has been reported to be greatly enhanced by PIP3, whereas that of the ß- or {delta}-PLC isozymes remains unchanged.26 Furthermore, overexpression of PI3K in COS-7 cells causes increased activation of PLC{gamma}1 that is reduced to the normal level when the cells are treated with PI3K inhibitors. In other studies, the SH2 and PH domains of PLC{gamma}1 have been found to potently bind PIP3 generated by PI3K.19 27

To investigate whether EGF-induced PLC{gamma}1 activation in RCECs is dependent on concomitant activation of PI3K, PLC{gamma}1 activity was assayed in cells treated with the PI3K inhibitors wortmannin and LY294002. The results showed that both wortmannin and LY294002 dose-dependently inhibited EGF-stimulated PLC{gamma}1 activation and cell proliferation (Figs. 5 6) . Because the cell number remained essentially unchanged with increasing concentrations of wortmannin or LY294002, it was unlikely that the inhibitory effects of these compounds on EGF-induced responses was due to increased cell death. When wortmannin or LY294002 was added directly to the PLC{gamma}1 assay mixture, there was no inhibition of the enzyme activity (data not shown), which suggests that these compounds inhibit PLC{gamma}1 by selectively blocking PI3K activity. Further evidence that PLC{gamma}1 lies downstream of PI3K is provided by the experiments in which PLC{gamma}1 was assayed in the presence of 3-phosphoinositides. The enzyme activity was markedly increased by PI(3,4,5)P3 >> PI(3,4)P2 and PI(3)P was without effect (Fig. 7) .

Taken together, the data provide support for the hypothesis that EGF-induced proliferation of RCECs involves both increased PLC{gamma}1 synthesis and enzyme activation by PI(3,4,5)P3, a product of PI3K. One likely mechanism for PLC{gamma}1 activation by PI(3,4,5)P3 may involve recruitment of the enzyme by PI(3,4,5)P3 to the plasma membrane adjacent to its substrate, PIP2. The SH2, SH3, and PH domains of PLC{gamma}1 can specifically interact with inositol phospholipids. After PLC{gamma}1 recruitment to the plasma membrane, the enzyme could be activated directly by PI(3,4,5)P3.

In conclusion, the current data demonstrate a close correlation between PLC{gamma}1 activation and cell proliferation in EGF-treated and untreated RCECs. PLC{gamma}1 activation involved both increased PLC{gamma}1 synthesis and tyrosine phosphorylation. Furthermore, the activity of PLC{gamma}1 was found to be modulated by PI3K, suggesting that PLC{gamma}1 lies downstream of PI3K. However, our studies did not provide information regarding the exact biochemical events that follow PLC{gamma}1 activation and culminate in increased DNA synthesis during cell proliferation. One second-messenger molecule generated by PIP2 hydrolysis is DAG, which is a natural activator of PKC. There are several studies suggesting that PKC can increase DNA synthesis by activating the Raf/mitogen-activated kinase kinase (MEK)/extracellular signal-regulated (ERK) pathway.28 29 Therefore, it is possible that the mitogen-activated protein (MAP) kinase cascade also plays a similar key role in EGF-stimulated wound repair in corneal epithelium.


   Acknowledgements
 
The authors thank Kaoru Araki-Sasaki for providing SV-40–transformed rabbit corneal epithelial cells and Rhea Markowitz for critical reading of the manuscript.


   Footnotes
 
Supported by Grant EY05738 from the National Institutes of Health.

Submitted for publication July 6, 2000; revised December 13, 2000 and February 8, 2001; accepted February 23, 2001.

Commercial relationships policy: N.

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: Rashid A. Akhtar, Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta, GA 30912-2100. raakhtar{at}mail.mcg.edu


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Berridge, M. (1993) Inositol phosphate and calcium signaling Nature 361,315-325[Medline][Order article via Infotrieve]
  2. Rhee, SG, Bae, YS (1997) Regulation of phosphoinositide specific phospholipase C isoforms J Biol Chem 272,15045-15048[Free Full Text]
  3. Toker, A, Cantley, LC (1997) Signaling through the lipid products of phosphoinositide-3-OH kinase Nature 387,673-676[Medline][Order article via Infotrieve]
  4. Wymann, MP, Pirola, L. (1998) Structure and function of phosphoinositide 3-kinase Biochim Biophys Acta 1436,127-150[Medline][Order article via Infotrieve]
  5. Ching, TT, Wang, D-S, Hsu, A-L, Lu, P-J, Chen, C-S. (1999) Identification of multiple phosphoinositide-specific phospholipases D as new regulatory enzymes for phosphatidylinositol 3,4,5-trisphosphate J Biol Chem 274,8611-8617[Abstract/Free Full Text]
  6. van der Geer, P, Hunter, T, Lindberg, RA (1994) Receptor protein-tyrosine kinases and their signal transduction pathways Ann Rev Cell Biol 10,251-337
  7. Reim, M, Busse, S, Leber, M, Schulz, C. (1988) Effect of epidermal growth factor in severe experimental alkali burns Ophthalmic Res 20,327-331[Medline][Order article via Infotrieve]
  8. Zhang, Y, Liou, GI, Gulati, AK, Akhtar, RA (1999) Expression of phosphatidylinositol 3-kinase during EGF-stimulated wound repair in rabbit corneal epithelium Invest Ophthalmol Vis Sci 40,2819-2826[Abstract/Free Full Text]
  9. Tao, W, Liou, GI, Wu, X, Reinach, PS (1995) ETB and epidermal growth factor receptor stimulation of wound closure in bovine corneal epithelial cells Invest Ophthalmol Vis Sci 36,2614-2622[Abstract/Free Full Text]
  10. Zhang, Y, Akhtar, RA (1997) Epidermal growth factor stimulation of phosphatidylinositol 3-kinase during wound closure in rabbit corneal epithelial cells Invest Ophthalmol Vis Sci 38,1139-1148[Abstract/Free Full Text]
  11. Islam, M, Akhtar, RA (2000) Epidermal growth factor stimulates phospholipase C{gamma}1 in cultured rabbit corneal epithelial cells Exp Eye Res 70,261-269[Medline][Order article via Infotrieve]
  12. Zhang, Y, Akhtar, RA (1996) Effect of epidermal growth factor on phosphatidylinositol 3-kinase activity in rabbit corneal epithelial cells Exp Eye Res 63,265-275[Medline][Order article via Infotrieve]
  13. Araki, K, Ohashi, Y, Sasabe, T, et al (1993) Immortalization of rabbit corneal epithelial cells by a recombinant SV40-adenovirus vector Invest Ophthalmol Vis Sci 34,2665-2671[Abstract/Free Full Text]
  14. Zhang, Y, Araki-Sasaki, K, Honda, H, Akhtar, RA (1995) Effect of carbachol on phospholipase C-mediated phosphatidylinositol 4,5-bisphosphate hydrolysis, and its modulation by isoproterenol in rabbit corneal epithelial cells Curr Eye Res 14,563-571[Medline][Order article via Infotrieve]
  15. Lowry, OH, Rosebrough, NJ, Farr, AL, Randal, RJ (1951) Protein measurements with Folin phenol reagent J Biol Chem 193,265-275[Free Full Text]
  16. Zhou, CJ, Akhtar, RA, Abdel-Latif, AA (1993) Purification and characterization of phosphoinositide-specific phospholipase C from bovine iris sphincter smooth muscle Biochem J 289,401-409
  17. Smith, MR, Liu, YL, Kim, H, Rhee, SG, Kung, HF (1990) Inhibition of serum- and Ras-stimulated DNA synthesis by antibodies to phospholipase C Science 247,1074-1077[Abstract/Free Full Text]
  18. Roche, S, McGlade, J, Jones, M, Gish, GD, Pawson, T, Courtneidge, SA (1996) Requirement of phospholipase C{gamma}, the tyrosine phosphatase Syp and the adaptor proteins Shc and Nck for PDGF-induced DNA synthesis: evidence for Ras-independent pathways EMBO J 15,4940-4948[Medline][Order article via Infotrieve]
  19. Wang, Z, Gluck, S, Zhang, L, Moran, MF (1998) Requirement for phospholipase C-{gamma}1 enzymatic activity in growth factor-induced mitogenesis Mol Cell Biol 18,590-597[Abstract/Free Full Text]
  20. Tatrai, A, Lee, SK, Stern, PH (1994) U-73122, a phospholipase C antagonist, inhibits effects of endothelin-1 and parathyroid hormone on signal transduction in UMR-106 osteoblastic cells Biochim Biophys Acta 1224,575-582[Medline][Order article via Infotrieve]
  21. Karlsson, S, Ahren, B. (1996) Gastrin-releasing peptide mobilizes calcium from intracellular stores in HIT-T15 cells Peptides 17,909-916[Medline][Order article via Infotrieve]
  22. Homma, Y, Takenawa, T. (1994) Inhibitory effect of src homology (SH)2/SH3 fragments of phospholipase C{gamma} on the catalytic activity of phospholipase C isoforms: identification of a novel phospholipase C inhibitor region J Biol Chem 267,21844-21849[Abstract/Free Full Text]
  23. Homma, MK, Yamasaki, M, Ohmi, S, Homma, Y. (1997) Inhibition of phosphoinositide hydrolysis and cell growth of Swiss 3T3 cells by myristoylated phospholipase C inhibitor peptides J Biochem 122,738-742[Abstract/Free Full Text]
  24. Sekiya, F, Bae, YS, Rhee, SG (1999) Regulation of phospholipase C isozymes: activation of phospholipase C-{gamma} in the absence of tyrosine-phosphorylation Chem Phys Lipids 98,3-11[Medline][Order article via Infotrieve]
  25. Kim, HK, Kim, JW, Zilberstein, A, et al (1991) PDGF stimulation of inositol phospholipid hydrolysis requires PLC{gamma}1 phosphorylation on tyrosine residues 783 and 1254 Cell 65,435-441[Medline][Order article via Infotrieve]
  26. Bae, YS, Cantley, LG, Chen, C-S, Kim, S-R, Kwon, K-S, Rhee, SG (1998) Activation of phospholipase C-{gamma} by phosphatidylinositol 3,4,5-trisphosphate J Biol Chem 273,4465-4469[Abstract/Free Full Text]
  27. Falasca, M, Logan, SK, Lehto, VP, Baccante, G, Lemmon, MA, Schlessinger, J. (1998) Activation of phospholipase C {gamma} by PI 3-kinase-induced PH domain-mediated membrane targeting EMBO J 17,414-422[Medline][Order article via Infotrieve]
  28. Force, T, Bonventre, JV (1998) Growth factors and mitogen activated protein kinases Hypertension 18,6983-6994
  29. Derkinderen, P, Enslen, H, Girault, J-A. (1999) The ERK/MAP-kinases cascade in the nervous system Neuroreport 10,R24-R34[Medline][Order article via Infotrieve]



This article has been cited by other articles:


Home page
 IOVSHome page
Z. Wang, H. Yang, S. D. Tachado, J. E. Capo-Aponte, V. N. Bildin, H. Koziel, and P. S. Reinach
Phosphatase-Mediated Crosstalk Control of ERK and p38 MAPK Signaling in Corneal Epithelial Cells
Invest. Ophthalmol. Vis. Sci., December 1, 2006; 47(12): 5267 - 5275.
[Abstract] [Full Text] [PDF]

Home page
 IOVSHome page
F. Zhang, Q. Wen, S. Mergler, H. Yang, Z. Wang, V. N. Bildin, and P. S. Reinach
PKC Isoform-Specific Enhancement of Capacitative Calcium Entry in Human Corneal Epithelial Cells.
Invest. Ophthalmol. Vis. Sci., September 1, 2006; 47(9): 3989 - 4000.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
T. Li and L. Lu
Epidermal Growth Factor-induced Proliferation Requires Down-regulation of Pax6 in Corneal Epithelial Cells
J. Biol. Chem., April 1, 2005; 280(13): 12988 - 12995.
[Abstract] [Full Text] [PDF]

Home page
 IOVSHome page
S. Landreville, S. Coulombe, P. Carrier, M. H. Gelb, S. L. Guerin, and C. Salesse
Expression of Phospholipases A2 and C in Human Corneal Epithelial Cells
Invest. Ophthalmol. Vis. Sci., November 1, 2004; 45(11): 3997 - 4003.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Islam, M.
Right arrow Articles by Akhtar, R. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Islam, M.
Right arrow Articles by Akhtar, R. A.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS