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. 2005 Jul;16(7):3236–3246. doi: 10.1091/mbc.E05-02-0109

Calcium-induced Human Keratinocyte Differentiation Requires src- and fyn-mediated Phosphatidylinositol 3-Kinase–dependent Activation of Phospholipase C-γ1

Zhongjian Xie 1, Patrick A Singleton 1, Lilly YW Bourguignon 1, Daniel D Bikle 1
Editor: M Bishr Omary1
PMCID: PMC1165407  PMID: 15872086

Abstract

We have previously demonstrated that phospholipase C (PLC)-γ1 is required for calcium-induced human keratinocyte differentiation. In the present study, we investigated whether the activation of PLC-γ1 by nonreceptor kinases such as src and fyn plays a role in mediating this process. Our results showed that the combination of dominant negative src and fyn blocked calcium-stimulated PLC-γ1 activity and human keratinocyte differentiation, whereas each separately has little effect. However, unlike the activation of PLC-γ1 by epidermal growth factor, calcium-induced activation of PLC-γ1 was not a result of direct tyrosine phosphorylation. Therefore, we examined an alternative mechanism, in particular phosphatidylinositol 3,4,5-triphosphate (PIP3) formed as a product of phosphatidylinositol 3-kinase (PI3K) activity. PIP3 binds to and activates PLC-γ1. The combination of dominant negative src and fyn blocked calcium-induced tyrosine phosphorylation of the regulatory subunit of PI3K, p85α, and the activity of the catalytic subunit of PI3K. PI3K inhibitors blocked calcium activation of PLC-γ1 as well as the induction of keratinocyte differentiation markers involucrin and transglutaminase. These data indicate that calcium activates PLC-γ1 via increased PIP3 formation mediated by c-src– and fyn-activated PI3K. This activation is required for calcium-induced human keratinocyte differentiation.

INTRODUCTION

Phospholipase C (PLC) catalyzes the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) to inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). These molecules regulate the mobilization of intracellular Ca2+ and protein kinase C activation, respectively (Berridge and Irvine, 1984; Majerus et al., 1986). To date, four types of phosphoinositide-specific PLC isozymes have been identified: PLC-β, PLC-γ, PLC-δ, and PLC-ε. Each type contains several subtypes (Rhee et al., 1989). All PLC family members contain X and Y domains that fold together to form the catalytic site (Rhee and Bae, 1997). In the γ-type isozymes, including PLC-γ1 and PLC-γ2, the linker region between the X and Y domains is extended and contains two Src homology (SH)2 domains and one SH3 domain (Carpenter and Ji, 1999). These two SH2 domains bind to phosphorylated tyrosine residues of receptor kinases including the epidermal growth factor (EGF) receptor, platelet-derived growth factor receptor, and nerve growth factor receptor, resulting in the tyrosine phosphorylation of PLC-γ isozymes (Carpenter and Ji, 1999). PLC-γ isozymes also are phosphorylated and activated by various nonreceptor tyrosine kinases, including src, fyn, lck, lyn, and hck (Liao et al., 1993; Ozdener et al., 2002; Boudot et al., 2003). Some of these kinases such as src and fyn are increased during human or mouse keratinocyte differentiation induced by calcium (Zhao et al., 1992; Calautti et al., 1995) and also control mouse keratinocyte cell-cell adhesion (Calautti et al., 1998, 2002).

PLC-γ isozymes also can be activated directly by several lipid-derived second messengers, including phosphatidic acid (Jones and Carpenter, 1993a), arachidonic acid (Hwang et al., 1996; Sekiya et al., 1999), and phosphatidylinositol 3,4,5-triphosphate (PIP3) (Bae et al., 1998; Falasca et al., 1998; Rameh et al., 1998) in the absence of tyrosine phosphorylation. Phosphatidic acid and arachidonic acid are primarily derived from phosphatidylcholine by the action of phospholipase D and phospholipase A2, respectively. PIP3 is primarily derived from PIP2 by the action of phosphatidylinositol 3-kinase (PI3K). Therefore, activation of PLC-γ isozymes may occur secondarily to the activation of phospholipase D, phospholipase A2, or PI3K.

Unlike PLC-γ2 expressed mainly in cells of hematopoietic origin, PLC-γ1 is found in a variety of cells, including keratinocytes. PLC-γ1 is required for calcium-induced human keratinocyte differentiation as we demonstrated previously (Xie and Bikle, 1999). In the present study, we investigated the mechanism by which PLC-γ1 is activated by calcium in human keratinocytes. We found that calcium induces src and fyn activity, that src and fyn activity are required for calcium activation of PLC-γ1, but that PLC-γ1 activation occurs through a mechanism that does not involve direct tyrosine phosphorylation of PLC-γ1. Rather, this mechanism involves PI3K activation and PIP3 generation. Inhibition of this mechanism blocks calcium-induced human keratinocyte differentiation.

MATERIALS AND METHODS

Cell Culture

Human keratinocytes were isolated from neonatal human foreskins and grown in serum-free keratinocyte growth medium (KGM; Cambrex Bio Science Walkersville, Walkersville, MD) as described previously (Pillai et al., 1988). Briefly, human keratinocytes were isolated from newborn human foreskins by trypsinization (0.25% trypsin, 4°C, overnight), and primary cultures were established in KGM containing 0.07 mM calcium. The second passage human keratinocytes were plated with KGM containing 0.03 mM calcium and used in the subsequent experiments. To initiate differentiation, the calcium concentration was raised to 1.2 mM.

Dominant Negative Constructs Transfection and Selection

The dominant negative src and fyn constructs were kindly provided by Dr. Filippo G. Giancotti (Memorial Sloan-Kettering Cancer Center, New York, NY). The kinase-inactivating mutation in fyn was a replacement of Lys 299 by Met, and in src it was a replacement of Lys 295 by Met. These dominant negative constructs have been shown to specifically inactivate src and fyn activities (Twamley-Stein et al., 1993). The dominant negative src and fyn inserts were resubcloned into pcDNA 3.1(–) (Invitrogen, Carlsbad, CA). Second passage human keratinocytes were transfected with dominant negative constructs by using TransIt-keratinocyte transfection reagent (Mirus, PanVera, Madison, WI). To enrich transfected cells, the transiently transfected cells were selected by a 2-d incubation with G418 at a concentration of 200 μg/ml starting 2 d after transfection as described previously (Xie and Bikle, 1999).

Cell Lysate Preparation, Western Analysis, and Immunoprecipitation

Human keratinocytes in cell culture plates were washed twice with phosphate-buffered saline (PBS) and then incubated in PBS containing 1% NP-40 and Complete protease inhibitors (Roche Diagnostics, Indianapolis, IN) or radioimmunoprecipitation assay (RIPA) lysis buffer containing 50 mM HEPES, pH 7.4, 1% Triton X-100, 0.1% SDS, 150 mM NaCl, 1 mM EDTA, and Complete protease inhibitors (Roche Diagnostics) for 5 min. Cells were scraped into Microfuge tubes, incubated at 4°C for 30 min, and pelleted by centrifugation. The supernatant was collected. The protein concentration of the lysate was measured by a bicinchoninic acid (BCA) protein assay kit (Pierce Chemical, Rockford, IL). Equal amounts of protein were electrophoresed through reducing SDS-PAGE and electroblotted onto polyvinylidene fluoride (PVDF) microporous membranes (Immobilon-P, 0.45 μm; Millipore, Billerica, MA). After incubation in blocking buffer (100 mM Tris base, 150 mM NaCl, 5% nonfat milk or 2% BSA, and 0.5% Tween 20), the blot was incubated overnight at 4°C with appropriate primary antibodies: monoclonal antibodies against human involucrin (Sigma-Aldrich, St. Louis, MO) at a dilution of 1:2000, monoclonal antibodies against human transglutaminase (Biomedical Technologies, Stoughton, MA) at a dilution of 1:200, polyclonal antibodies against PLC-γ1 (Santa Cruz Biotechnology, Santa Cruz, CA) at a dilution of 1:200, polyclonal antibodies against phospho-PLC-γ1 (Tyr 783; Santa Cruz Biotechnology) at a dilution of 1:200, polyclonal antibodies against phospho-PLC-γ1 (Tyr 1254; Santa Cruz Biotechnology) at a dilution of 1:200, monoclonal antibodies against phosphotyrosine-containing proteins (PY99; Santa Cruz Biotechnology) at a dilution of 1:200, polyclonal antibodies against src and fyn (Santa Cruz Biotechnology) at a dilution of 1:200, polyclonal antibodies against Akt and phosphorylated Akt (Cell Signaling Technology, Beverly, MA) at a dilution of 1:1000, and polyclonal antibodies against PI3K p85α (Santa Cruz Biotechnology) at a dilution of 1:200. After washes in the blocking buffer, the membranes were incubated for 1 h with the appropriate anti-IgG secondary antibody conjugated to horseradish peroxidase (Amersham Biosciences. Piscataway, NJ) in the blocking buffer. After a second series of washes, bound antibody complexes were visualized using the SuperSignal ULTRA chemiluminescent kit (Pierce Chemical) and subsequent exposure to x-ray film. In some experiments, equal amounts of protein were incubated with a specific antibody at 4°C for 1 h and then with UltraLink immobilized protein G (Pierce Chemical) at 4°C overnight. Primary antibodies included monoclonal antibodies against phosphotyrosine (p-Tyr; Santa Cruz Biotechnology) and polyclonal antibodies against PLC-γ1 (Santa Cruz Biotechnology). The lysate-antibody-agarose beads mixture was washed four times with PBS and then analyzed by Western analysis as described above.

PLC-γ1 Activity Assay

PLC-γ1 activity was determined by measuring accumulation of IP3 according to the experimental procedure described recently (Bourguignon et al., 2004). Cells in 150-mm dishes were washed with PBS containing 0.1% sodium orthovanadate and 0.1% NaF and then incubated with RIPA lysis buffer or 1% NP-40 containing 0.1% sodium orthovanadate, 0.1% NaF, and Complete protease inhibitors (Roche Diagnostics) for 5 min. Cells were scraped into Microfuge tubes and incubated at 4°C on a rotator for 1 h. The supernatant collected after centrifugation was chromatographed over a wheat germ agglutinin column (EY Laboratories, San Mateo, CA). The eluent containing 100 μg of protein was incubated with 2 μg polyclonal PLC-γ1 antibodies (BD Biosciences, San Jose, CA) at 4°C overnight and then 20 μl of UltraLink immobilized protein G (Pierce Chemical) at 4°C for 1 h. After centrifugation, the pellet was washed with reaction buffer containing 10 mM HEPES, pH 7.0, 10 mM NaCl, 120 mM KCl, 2 mM EGTA, 0.05% deoxycholate, 5 μg/ml BSA, and 10 μM CaCl2 and resuspended in 800 μl of reaction buffer. In triplicates, 50 μl of suspension was incubated with 10 μM [3H]PIP2 (6.5 Ci/mmol; PerkinElmer Life and Analytical Sciences, Boston, MA), phosphatidylcholine (Sigma-Aldrich), and phosphatidylserine (Sigma-Aldrich) in a molar ratio of 1:3:3 in the reaction buffer. The reaction was ended by adding 200 μl of 10% trichloroacetic acid (TCA) at 1, 2, 3, 4, and 5 min and then 200 μl of 10% BSA. The radioactivity of 50 μl of supernatant after centrifugation was determined by a scintillation counter. The concentration of [3H]IP3 produced was calculated based on the specific activity of the substrate.

src, fyn, and lyn Kinase Activity Assays

src kinase assays were carried out using the src assay kit according to the manufacturer's instructions (Upstate Biotechnology, Charlottesville, VA). fyn and lyn kinase assays were carried out according to a protocol modified based on the instructions for the src kinase assay. Briefly, 0.5 mg of total cellular protein isolated from human keratinocytes in 1 ml of buffer A [50 mM Tris-HCl, pH 7.5, 1 mM EGTA, 1 mM EDTA, 1 mM activated sodium orthovanadate, 10 mM sodium β-glycerol phosphate, 50 mM NaF, 5 mM sodium pyrophosphate, 0.1% (wt/vol) Triton X-100, 0.1% 2-mercaptoethanol, 0.27 M sucrose, and 1 μM microcystin LR] containing Complete protease inhibitors (Roche Diagnostics) was incubated with 2 μg of src polyclonal antibody, 2 μg of fyn polyclonal antibody, or 2 μg of lyn polyclonal antibody at 4°C for 1 h. Immunocomplexes were collected by incubation with 20 μl of UltraLink immobilized protein G (Pierce Chemical) overnight. For the src activity assay, the immunoprecipitate was washed three times with ice-cold buffer A and then incubated with 10 μl (150 μM final concentration) of the substrate peptide, 10 μl of src reaction buffer, and 10 μl (0.01 μCi) of [γ-32P]ATP (Amersham Biosciences) in a total volume of 40 μl for 30 min at 30°C with agitation. To precipitate the peptide, 20 μl of 40% TCA was added and incubated for 5 min at room temperature. In triplicate, a 25-μl aliquot was transferred onto the center of a numbered P81 paper square. The assay squares were washed five times for 5 min each with 0.75% phosphoric acid and once with acetone. The assay squares were transferred to a scintillation vial, 10 ml of scintillation cocktail added, and the level of radioactivity was determined by a scintillation counter. For the fyn or lyn activity assay, the immunoprecipitate was washed three times with fyn/lyn assay buffer and then incubated with 2.5 μl (250 μM final concentration) of the appropriate substrate peptide, 6.25 μl of assay buffer, and 10 μl (0.01 μCi) of [γ-32P]ATP (Amersham Biosciences) in a total volume of 25 μl for 30 min at 30°C with agitation. In triplicate, a 6-μl aliquot was transferred onto the center of a numbered P81 paper square. The assay squares were washed three times for 5 min each with 0.75% phosphoric acid and once with acetone for 1 min. The assay squares were transferred to a scintillation vial, 10 ml of scintillation cocktail added, and the level of radioactivity was determined by a scintillation counter.

PI3K Activity Assay

The PI3K activity assay was performed according to the experimental procedure described recently (Bourguignon et al., 2003). Briefly, cells were washed with PBS and solubilized in buffer A (1% NP-40, 1% sodium orthovanadate, 1% phenylmethylsulfonyl fluoride [PMSF], and 25 ng/ml okadaic acid) containing Complete protease inhibitors (Roche Diagnostics) at 4°C for 1 h followed by immunoprecipitation with antibody-conjugated beads against p110α, p110β, p110γ, or p110δ. The antibody conjugated beads were then incubated in 50 μl of buffer A containing 20 μg of sonicated PIP2 (Sigma-Aldrich) and 10 μCi of [γ-32P]ATP for 30 min at 32°C. The total ATP concentration in the reaction was 1 mM. Subsequently, the reaction was terminated by the addition of acidified chloroform:methanol [1:1 (vol/vol)]. Extracted lipids were then spotted and developed using a solvent system containing chloroform:acetone:methanol:acetic acid/water (80:30:26:24:14). The thin layer chromatography plates were dried, and radioactively labeled PIP3 spots were visualized by autoradiography.

PIP3 Assay

One hundred micrograms of total protein extracted from human keratinocytes with 1% NP-40 was incubated with PLC-γ1 antibody at 4°C overnight and then with UltraLink immobilized protein G (Pierce Chemical) for 1 h. PIP3 bound to PLC-γ1 was extracted by chloroform and then spotted onto a PVDF membrane. After incubation in blocking buffer (100 mM Tris base, 150 mM NaCl, 5% nonfat milk or 2% BSA, and 0.5% Tween 20), the membrane was incubated overnight at 4°C with a monoclonal antibody against PIP3 (Echelon Research Laboratories, Salt Lake City, UT) at a concentration of 1 μg/ml. After washes in the blocking buffer, the membranes were incubated for 1 h with rabbit anti-IgG secondary antibody conjugated to horseradish peroxidase (Amersham Biosciences) in the blocking buffer. After a second series of washes, bound antibody complexes were visualized using the Super-Signal ULTRA chemiluminescent kit (Pierce Chemical) and subsequent exposure to x-ray film.

RESULTS

Several members of the src family of tyrosine kinases are expressed in keratinocytes, including src and fyn, and each has been shown to be increased in protein levels and activities in human or mouse keratinocytes induced to differentiate by calcium (Zhao et al., 1992; Calautti et al., 1995). To confirm these results, human keratinocytes were treated with 1.2 mM calcium, and src and fyn activities in cells were determined and normalized to the protein level in the immunoprecipitates. We found that calcium increased the intrinsic activity and protein level of src above control in a time-dependent manner (Figure 1A). The induction of src activity by calcium occurred as early as 5 min, with a peak more than threefold increase by 30 min. The induction of src protein levels in the immunoprecipitates by calcium occurred as early as 1 h, with a plateau starting at 6 h (Figure 1A). The induction of the intrinsic activity of fyn by calcium also occurred as early as 5 min (Figure 1B). However, fyn protein levels in the immunoprecipitates were not induced by calcium (Figure 1B). Similar data were obtained when total cellular extracts of src and fyn were analyzed for protein levels (our unpublished data). These results demonstrate that calcium induces both activity and protein levels of src but only activity of fyn. To investigate the role of src family tyrosine kinases in human keratinocyte differentiation, human keratinocytes were pretreated with a specific src family inhibitor PP2 for 1 h, and the responses of keratinocyte differentiation markers involucrin and transglutaminase to 1.2 mM calcium were examined (Figure 2A). The results showed that the levels of involucrin and transglutaminase were induced >3- and 12-fold, respectively, with 24 h of calcium administration. The induction of involucrin by calcium was reduced over threefold with PP2 treatment. The level of transglutaminase in PP2-treated keratinocytes was reduced to a level undetectable by Western analysis. In contrast, calcium-induced involucrin and transglutaminase were not affected by the vehicle control (dimethyl sulfoxide, DMSO) and an inactive analogue PP3 (Figure 2A). These results indicate that the src family is critical in calcium-induced human keratinocyte differentiation. To identify the isoforms of the src family involved in human keratinocyte differentiation, we transfected src and fyn dominant negative constructs into human keratinocytes. To enrich the fraction of transfected cells, cells were then treated with neomycin (G418) for 2 d. To determine the transfection efficiency, in pilot experiments, we first transfected an enhanced green fluorescent protein (EGFP)-expression vector into human keratinocytes at a density of 40% confluence. Two days later, cell density reached 85 to 90% confluence, and the EGFP was expressed in ∼40% of the cell population. To determine the efficiency of G418, killing curves of nontransfected keratinocytes by G418 were established in pilot experiments by treating 100% confluent keratinocyte cultures with different doses of G418 (50–400 μg/ml) for various lengths of time (1–5 d). We found that treatment with 200 μg/ml G418 for 2 d consistently killed >85% of the cells without affecting cell differentiation. Using this transfection method, equal amounts of dominant negative constructs for src and fyn or the combination of both were transfected into human keratinocytes, and the cells were then treated with 1.2 mM calcium for 24 h after a 2-d G418 selection. The results showed that neither dominant negative src nor fyn alone affected the calcium induction of involucrin and transglutaminase. However, the combination of dominant negative src and fyn completely blocked this induction (Figure 2B). The expression levels of dominant negative src and fyn in human keratinocytes were determined by Western analysis by using src and fyn antibodies. Although calcium increased endogenous src levels, it did not alter the expression of the dominant negative constructs. Because the total amount of DNA transfected into these cells was the same for the combination of dominant negative src and fyn as when transfected individually, the expression of either src or fyn in the combined transfection was ∼50% of that for either construct transfected by itself (Figure 2B). To determine the specificity of src and fyn dominant negative constructs, the activities for src, fyn, and lyn were evaluated. The activity of lyn was used as a negative control for the inhibitory effect of dominant negative src and fyn constructs. The results showed that the src or fyn dominant negative construct specifically inhibited src or fyn activity. Neither the src nor fyn dominant negative construct affected lyn activity. The data indicate that the effects of both src and fyn dominant negative constructs are specific (Figure 2C).

Figure 1.

Figure 1.

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Calcium stimulates src and fyn activities. Cultured human keratinocytes were treated with 1.2 mM calcium. The cells were harvested at the time points indicated. The activities of src (A) and fyn (B) were assayed as described in Materials and Methods and normalized to the relative protein levels in the immunoprecipitates. The relative activities are expressed as the percentage of that at zero time point. Protein levels of c-src (A) and fyn (B) in the immunoprecipitates were measured by Western analysis.

Figure 2.

Figure 2.

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The src family kinase inhibitor PP2 and the combination of dominant negative src and fyn block calcium-induced involucrin and transglutaminase expression. (A) Cultured human keratinocytes were treated with a specific src family kinase inhibitor (PP2) or its inactive analogue (PP3) at a concentration of 10 μM for 1 h. Cells were then stimulated with calcium for 24 h, and cells were harvested. The protein levels of involucrin and transglutaminase were determined by Western analysis. (B and C) Human keratinocytes cultured in KGM containing 0.03 mM calcium were transfected with dominant negative src (src-DN) and/or fyn (fyn-DN). Cells in each plate contained equal amounts of DNA during the transfection. Cells were then stimulated with 1.2 mM calcium for 24 h, and cells were harvested. The protein levels for involucrin, transglutaminase, src, and fyn were measured by Western analysis (B). The activities of src, fyn, and lyn (a control for determining the specificity of src-DN and fyn-DN) were assayed as described in Materials and Methods and are expressed as the percentage of the vector control at 0.03 mM calcium (C).

To determine whether the calcium-induced src and fyn activities are associated with activation of PLC-γ1, human keratinocytes were treated with 1.2 mM calcium, cells were then harvested after 5, 15, and 30 min and 1 h, and PLC-γ1 activity was measured (Figure 3). Activity was assessed by the level of IP3 production. The results showed that stimulation of PLC-γ1 activity occurred as early as 15 min after calcium administration. One-hour calcium treatment stimulated PLC-γ1 activity by up to 14-fold (Figure 3). To determine whether the activation of PLC-γ1 by calcium requires src kinases, human keratinocytes were pretreated with the src family inhibitor PP2 before the administration of calcium for 1 h. PLC-γ1 activity was determined 1 h later (Figure 4A). The results showed that calcium-stimulated PLC-γ1 activity was reduced by fivefold by PP2 (Figure 4A). To determine whether calcium-activated src and fyn contribute to the activation of PLC-γ1, human keratinocytes were transfected with dominant negative src and fyn or the combination of both (Figure 4B). PLC-γ1 activity in the cells was measured. The results showed that calcium stimulated PLC-γ1 activity by eightfold in cells transfected with the vector pcDNA3.1. Transfection either with dominant negative src or dominant negative fyn had little or no effect on calcium-stimulated PLC-γ1 activity, but the combination of dominant negative src and fyn reduced calcium-induced PLC-γ1 activity more than threefold (Figure 4B). These data indicate that both src and fyn are required for the activation of PLC-γ1 by calcium. To test whether the induced PLC-γ1 activity by calcium is due to tyrosine phosphorylation of PLC-γ1 by src and fyn, the effect of calcium on the levels of PLC-γ1 phosphorylated at Tyr 783 and Tyr 1254 were determined (Figure 5). These sites are known to be required for the activation of PLC-γ1. The results showed that neither of these two phosphorylation sites of PLC-γ1 was regulated by calcium (Figure 5). Immunoprecipitation with either anti-PLC-γ1 or anti-p-Tyr followed by Western with either anti-p-Tyr or anti-PLC-γ1 showed no stimulation of any tyrosine phosphorylation in PLC-γ1 by calcium (Figure 5). As a positive control, EGF treatment stimulated tyrosine phosphorylation of PLC-γ1 within 5 min (Figure 5). These data indicate that calcium induces PLC-γ1 activity through src and fyn by a mechanism that does not involve tyrosine phosphorylation of PLC-γ1. To explore other mechanisms, we tested whether PIP3 could activate PLC-γ1 as has been reported in other systems. Immunoprecipitated PLC-γ1 in human keratinocytes was incubated with PIP3, and then PLC-γ1 activity was assayed. The results showed that PIP3 induced PLC-γ1 activity (Figure 6A). To determine whether endogenous PIP3 remains bound to PLC-γ1 after immunoprecipitation and whether this binding is increased by calcium, human keratinocytes were treated with 1.2 mM calcium and lysed in 1% NP-40. PLC-γ1 was then immunoprecipitated by PLC-γ1 antibody. PIP3 in the immunoprecipitate was then measured by PIP3 antibody. The results showed that calcium increases the amount of PIP3 bound to PLC-γ1 even after lysis in 1% NP-40 and immunoprecipitation, supporting the conclusion that the increased PLC-γ1 activity after calcium administration is due to its stimulation by PIP3 (Figure 6B).

Figure 3.

Figure 3.

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Calcium stimulates PLC-γ1 activity. Cultured human keratinocytes were treated with 1.2 mM calcium. The cells were harvested at the time points indicated after the addition of calcium. The activity of PLC-γ1 was assayed as described in Materials and Methods by using a time course from zero to 15 min for each reaction. The activity of PLC-γ1 was expressed as picomoles of IP3 formed and normalized to the total protein content used for the immunoprecipitation.

Figure 4.

Figure 4.

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The src family kinase inhibitor PP2 and the combination of dominant negative src and fyn reduce calcium-stimulated PLC-γ1 activity. (A) Cultured human keratinocytes were treated with a specific src family kinase inhibitor (PP2) or its inactive analogue (PP3) for 1 h. Cells were then stimulated with calcium for 1 h, and cells were harvested. The PLC-γ1 activity was assayed as described in Materials and Methods and expressed as the percentage of the vehicle control at 0.03 mM calcium. (B) Human keratinocytes cultured in KGM containing 0.03 mM calcium were transfected with dominant negative src (src-DN) and/or fyn (fyn-DN). Equal amounts of DNA were added to cells in each plate during the transfection. Cells were then stimulated with calcium for 1 h, and cells were harvested. The PLC-γ1 activity was assayed as described in Materials and Methods and expressed as the percentage of the vector control at 0.03 mM calcium.

Figure 5.

Figure 5.

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Calcium does not stimulate the phosphorylation of PLC-γ1. Cultured human keratinocytes were treated with 1.2 mM calcium or 50 ng/ml EGF. The cells were harvested at the time points indicated. The protein levels of PLC-γ1 and its phosphorylated forms (p-PLC-γ1-pY783 and p-PLC-γ1-pY1254) were analyzed by Western analysis. In addition, the total phospho-PLC-γ1 was immunoprecipitated by antibodies against phosphotyrosine (p-Tyr) or PLC-γ1 followed by Western analysis with antibodies against PLC-γ1 or p-Tyr.

Figure 6.

Figure 6.

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PIP3 stimulates PLC-γ1 activity, and the binding of PIP3 to PLC-γ1 is induced by calcium. (A). Human keratinocytes were cultured in KGM with 0.03 mM calcium. The total cell lysate was isolated with the cell lysis buffer containing 50 mM HEPES, pH 7.5, 150 mM NaCl, 20 mM MgCl2, 1.0% NP-40, 0.4 mM Na3VO4, 40 mM NaF, 50 μM okadaic acid, 0.2 mM PMSF, and Complete protease inhibitors (Roche Diagnostics). PLC-γ1 in the lysate was immunoprecipitated with PLC-γ1 antibody. The immunoprecipitate was incubated with PIP3 (Sigma-Aldrich) dissolved in the cell lysis buffer at a final concentration of 1 μM for 1 h. PLC-γ1 activity was assayed as described in Materials and Methods and expressed as the percentage of the activity in the absence of PIP3 with 1 min [3H]PIP2 incubation. (B). The total cell lysate was isolated from human keratinocytes treated with or without calcium for 1 h, and PLC-γ1 in the lysate was immunoprecipitated with PLC-γ1 antibody. The immunoprecipitate was extracted with chloroform and then spotted onto a PVDF membrane. The amount of PIP3 on the membrane was determined by PIP3 antibody.

Because PI3K is a major means by which PIP3 is produced in cells, we next evaluated whether calcium could activate PI3K, and whether src kinases participated in that activation. We first wanted to know whether calcium regulates the phosphorylation of PI3K-p85α, a regulatory subunit of PI3K-p110α, β, and δ. The results showed that calcium increased the level of phospho-p85α in a time-dependent manner (Figure 7A). The increase was seen as early as 5 min and reached a peak threefold increase at 8 h. The total protein level of p85α was not altered by calcium. As a control, the protein level of involucrin was induced by calcium in a time-dependent manner (Figure 7A). The 1-h treatment with calcium also increased p110α, β, γ, and δ activity, although p110β activity was less stimulated than the others (Figure 7B). Phosphorylation of Akt is another downstream effect after PI3K activation. Therefore, we determined the effect of calcium on serine phosphorylation of Akt and found that calcium increases phosphorylated Akt for at least an hour (Figure 7C), correlating with the activation of PLC-γ1 by calcium, and confirming the activation of PI3K as assessed by PIP3 production.

Figure 7.

Figure 7.

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Calcium stimulates PI3K-p85α phosphorylation, PI3K activity, and Akt phosphorylation. (A). Cultured human keratinocytes were treated with 1.2 mM calcium for the indicated times. The protein levels of PI3K-p85α and its phosphorylated form (p-PI3K-p85α) were determined by Western analysis. Involucrin was used as a positive control for calcium induction (B). Human keratinocytes were treated with 1.2 mM calcium for 1 h. Cells were then harvested and PI3K-p110α, β, γ, and δ activities were measured as described in Materials and Methods (C). Cultured human keratinocytes were treated with 1.2 mM calcium for the indicated times. The protein levels of Akt and its phosphorylated form (p-Akt) were determined by Western analysis.

These data indicate that calcium activates p110α, β, and δ by inducing phosphorylation of p85α. Activation of p110γ is presumably by another mechanism in that it is controlled by the regulatory subunit p101 (Vanhaesebroeck and Waterfield, 1999). To determine whether src and fyn are responsible for the calcium-stimulated p85α phosphorylation, human keratinocytes were pretreated with PP2 for 1 h, and the response to calcium was examined (Figure 8A). The results showed that PP2 prevented the increase in phosphorylated p85α (p-p85α) by calcium, whereas treatment with vehicle (DMSO) or the inactive analogue PP3 had no effect (Figure 8A). To confirm these results, dominant negative constructs for src and fyn or the combination of both were then transfected into human keratinocytes, the cells were treated with 1.2 mM calcium, and the effect on p85α phosphorylation was measured (Figure 8B). The results showed that neither dominant negative src nor fyn alone affected calcium-induced phosphorylation of p85α, whereas the combination of dominant negative src and fyn blocked calcium-induced phosphorylation of p85α (Figure 8B). Furthermore, the combination of dominant negative src and fyn blocked the activation of p110α and δ and reduced the activation of γ (Figure 8C). In contrast, inhibition of PI3K activity by LY294002 affected neither src nor fyn activity. The inhibitive effect of LY294002 on the Akt phosphorylation induced by calcium was used as a positive control for the experiment (Figure 8D). These data suggest that both src and fyn are required for the activation of PI3K by calcium, whereas PI3K is not required for the activation of either src or fyn by calcium. Having shown that PIP3, a product of PI3K, stimulated PLC-γ1 activity, we then wanted to know whether the activation of PLC-γ1 requires PI3K activity. To address this issue, human keratinocytes were pretreated with LY294002 for 1 h followed by calcium for 1 h at which point PLC-γ1 activity was determined (Figure 9). The results showed that LY294002 blocked the induction of PLC-γ1 activity by calcium (Figure 9). These data indicate that PI3K is required for calcium-induced PLC-γ1 activation. To determine whether PI3K is involved in calcium-induced human keratinocyte differentiation, human keratinocytes were pretreated with LY294002 for 1 h before calcium administration, and the differentiation markers for keratinocyte differentiation, involucrin, and transglutaminase were examined 24 h later (Figure 10). The results showed that LY294002 prevented the induction of involucrin and transglutaminase by calcium (Figure 10). The data indicate that PI3K is required for calcium-induced human keratinocyte differentiation.

Figure 8.

Figure 8.

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The src kinase inhibitor and dominant negative src and fyn block calcium-stimulated PI3K-p85α phosphorylation and PI3K activity, whereas PI3K inhibition does not affect calcium stimulation of either src or fyn activity. Cultured human keratinocytes were preincubated for 1 h with PP2 or its inactive analog PP3 at a concentration of 10 μM (A), transfected with dominant negative src (src-DN), and/or fyn (fyn-DN), or vector alone (B and C). For the p85α phosphorylation (A and B) and p110α, β, γ, and δ activity (C) assay, transfected cells were then treated with 1.2 mM calcium for 1 h. The protein levels of the phosphorylated PI3K-p85α were determined by Western analysis. The activities of PI3K-p110α, β, γ, and δ were measured as described in Materials and Methods. (D) Cultured human keratinocytes were preincubated with 10 μM LY294002 or vehicle DMSO for 1 h. Cells were then stimulated with 1.2 mM calcium for 15 min, and cells were harvested. The src and fyn activities was assayed as described in Materials and Methods and expressed as the percentage of the vehicle control of the src activity. The levels of Akt phosphorylation and total Akt were assayed by Western analysis as described in Materials and Methods.

Figure 9.

Figure 9.

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PI3K inhibition blocks the stimulation of PLC-γ1 activity by calcium. Cultured human keratinocytes were preincubated with 10 μM LY294002 (+) or vehicle DMSO (–) for 1 h. Cells were then stimulated with 1.2 mM calcium for 1 h, and cells were harvested. The PLC-γ1 activity was assayed as described in Materials and Methods and expressed as the percentage of the activity at 0.03 mM calcium in the absence of LY294002.

Figure 10.

Figure 10.

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PI3K inhibition blocks the induction of involucrin and transglutaminase expression by calcium. Cultured human keratinocytes were preincubated with 10 mM LY294002 (+) or vehicle DMSO (–) for 1 h and then treated with 1.2 mM calcium for 24 h. The protein levels of involucrin and transglutaminase were determined by Western analysis.

DISCUSSION

In mouse keratinocyte cultures, IP3 and DAG levels increase within seconds to minutes after adding high calcium to the cells, suggesting an activation of the PLC pathway (Jaken and Yuspa, 1988; Tang et al., 1988; Moscat et al., 1989; Reiss et al., 1991). The increased IP3 production is thought to account for the initial rise in intracellular calcium by release of calcium from intracellular stores. However, the levels of inositol phosphates (IPs) remain elevated for hours after adding high calcium to mouse keratinocytes, indicating ongoing activation of the PLC pathway. This prolonged increase in IPs seems to be due to calcium activation of PLC-γ1 (Punnonen et al., 1993). Our data demonstrate that the activation of PLC-γ1 by calcium remains for at least 1 h after the calcium switch, consistent with previous findings. We have previously reported that PLC-γ1 is required for the calcium-induced increase in intracellular calcium and subsequent differentiation of human keratinocytes (Xie and Bikle, 1999). The present study shows for the first time that activation of PLC-γ1 by both src and fyn, but not src or fyn alone, is required for calcium-stimulated human keratinocyte differentiation. It was reported previously that fyn but not src is induced by calcium in mouse keratinocytes (Calautti et al., 1995). Mice lacking fyn display skin abnormalities, suggesting impaired epidermal differentiation (Ilic et al., 1997). However, in human keratinocytes, calcium induces c-src but not fyn (Zhao et al., 1992; current study). However, inhibition of both src and fyn activity and expression was required to block calcium-induced human keratinocyte differentiation, suggesting that both src and fyn are involved. PLC-γ1 is known to be tyrosine-phosphorylated by receptor kinases at a number of positions. The tyrosine phosphorylation sites on PLC-γ1 resulting from EGF stimulation have been mapped to Tyr 771 and Tyr 783, which sit between the C-SH2 and SH3 domains, and to Tyr 1254 in the C terminus (Kim et al., 1990; Nishibe et al., 1990). Mutagenesis indicates that the phosphorylation at Tyr 783 is essential for IP3 formation, phosphorylation of Tyr 771 is dispensable, and phosphorylation of Tyr 1254 is necessary to achieve maximal IP3 formation (Kim et al., 1991). In addition to the receptor kinases, src, fyn, lck, lyn, and hck kinases are all capable of phosphorylating PLC-γ1 in vitro (Liao et al., 1993; Lee and Rhee, 1995; Noh et al., 1995). However, it is not clear that any of the src family kinases are responsible for tyrosine phosphorylation of PLC-γ1 in vivo. Although tyrosine phosphorylation has a limited effect on the activity of PLC-γ1 in vitro, it has been reported to be essential for activation of the phospholipase in vivo (Kim et al., 1991). Data presented in this article indicate that both src and fyn are required for the calcium-induced PLC-γ1 activation, but this activation does not involve tyrosine phosphorylation of PLC-γ1. PLC-γ1 also is phosphorylated on serine and threonine in other cell types. However, the functional significance of such phosphorylation is unknown, and there is no evidence to suggest it alters enzyme activity (Kim et al., 1991). Furthermore, in studies not reported here, we have found that calcium does not affect serine phosphorylation of PLC-γ1 (our unpublished data).

Several studies indicate that PLC-γ1 can be activated by phosphatidic acid (Jones and Carpenter, 1993b), arachidonic acid (Hwang et al., 1996; Sekiya et al., 1999), and PIP3 (Bae et al., 1998; Falasca et al., 1998; Rameh et al., 1998) in the absence of tyrosine phosphorylation. These lipid-derived messengers are the immediate products of phospholipase D, phospholipase A2, and PI3K, respectively, and they provide alternative mechanisms to tyrosine phosphorylation by which PLC-γ1 can be activated. Recent observations indicate that src activates PI3K by an unknown mechanism in other cell types (Encinas et al., 2001; Kubota et al., 2001). Several classes of PI3K have been distinguished on the basis of sequence similarity and substrate selectivity (Toker and Cantley, 1997). The best-studied class Ia PI3Ks consist of a p110 catalytic subunit (p110α, β, or δ) and a tightly associated adapter (or regulatory) subunit encoded by at least three distinct genes (p85α, p85β, or p55γ) (Escobedo et al., 1991; Domin and Waterfield, 1997). This subunit has no known enzymatic activity but serves a regulatory function. Alternative splicing of the p85α gene results in the generation of two small splice variants, p55α and p50α, which also associate with p110 catalytic subunit isoforms. Class Ib PI3Ks contain a p110γ catalytic subunit, and they do not interact with p85 adaptor molecules but with a p101 regulatory subunit. Furthermore, they are directly activated by βγ subunits of G proteins rather than by kinases. The p85α is the most abundantly expressed regulatory subunit of PI3Ks. Our data indicate that in human keratinocytes the phosphorylation of p85α and the activation of PI3K α, β, and δ by calcium requires src and fyn. Mouse keratinocytes seem to have a different mechanism in the regulation of PI3K by calcium. Filvaroff et al. (1992) reported that in these cells the phosphorylation of p85α was not induced by calcium. Although 110γ does not associate with p85α, calcium activation of p110γ also requires src and fyn, suggesting other mechanisms are involved. Activated PI3K converts PIP2 into PIP3 at the plasma membrane. PIP3 binds to the N-terminal pleckstrin homology domain (Falasca et al., 1998) and the C-terminal SH2 domain (Bae et al., 1998; Rameh et al., 1998) of PLC-γ1 to target PLC-γ1 to the membrane. Our data demonstrate that PIP3 binds to and activates PLC-γ1, supporting our conclusion that PI3K is upstream of PLC-γ1 activation. Together, we conclude that in human keratinocytes PIP3 produced by src and fyn activated PI3K provides an “anchor” for PLC-γ1 at the membrane that is crucial for the positioning and activation of PLC-γ1 after calcium administration (Figure 11). Our data further indicate that this mechanism is required for calcium-induced human keratinocyte differentiation.

Figure 11.

Figure 11.

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Proposed model for the signaling pathway of PLC-γ1 activation mediating calcium-induced human keratinocyte differentiation. Calcium (Ca2+) binding to the calcium receptor (CaR) (Tu et al., 2001) stimulates src and fyn, which activate PI3K via phosphorylation of p85α leading to PIP3 accumulation. PIP3 and then stimulates PLC-γ1 activity, which increases IP3 production. Intracellular calcium ([Ca2+]i) release from endoplasmic reticulum (ER) and Golgi stores mediated by IP3 binding to the IP3 receptor (IP3R) triggers human keratinocyte differentiation.

This article was published online ahead of print in MBC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E05–02–0109) on May 4, 2005.

Abbreviations used: DAG, diacylglycerol; EGF, epidermal growth factor; IP3, inositol 1,4,5-trisphosphate; KGM, keratinocyte growth medium; PI3K, phosphatidylinositol 3-kinase; PIP2, phosphatidylinositol 4,5-bisphosphate; PIP3, phosphatidylinositol 3,4,5-bisphosphate; PLC, phospholipase C.

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