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J. Biol. Chem., Vol. 283, Issue 6, 3519-3528, February 8, 2008

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Inactivation of the Calcium Sensing Receptor Inhibits E-cadherin-mediated Cell-Cell Adhesion and Calcium-induced Differentiation in Human Epidermal Keratinocytes^*

Chia-Ling Tu¹, Wenhan Chang, Zhongjian Xie^¶, and Daniel D. Bikle

From the Endocrine Unit, Veteran Affairs Medical Center, ^¶Northern California Institute for Research and Education and University of California, San Francisco, California 94121

Received for publication, October 5, 2007 , and in revised form, November 12, 2007.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Extracellular Ca²⁺ (Ca²⁺_o) is a critical regulator that promotes differentiation in epidermal keratinocytes. The calcium sensing receptor (CaR) is essential for mediating Ca²⁺ signaling during Ca²⁺_o-induced differentiation. Inactivation of the endogenous CaR-encoding gene CASR by adenoviral expression of a CaR antisense cDNA inhibited the Ca²⁺_o-induced increase in intracellular free calcium (Ca²⁺_i) and expression of terminal differentiation genes, while promoting apoptosis. Ca²⁺_o also instigates E-cadherin-mediated cell-cell adhesion, which plays a critical role in orchestrating cellular signals mediating cell survival and differentiation. Raising Ca²⁺_o concentration ([Ca²⁺]_o) from 0.03 to 2 mM rapidly induced the co-localization of -, β-, and p120-catenin with E-cadherin in the intercellular adherens junctions (AJs). To assess whether CaR is required for the Ca²⁺_o-induced activation of E-cadherin signaling, we examined the impact of CaR inactivation on AJ formation. Decreased CaR expression suppressed the Ca²⁺_o-induced AJ formation, membrane translocation, and the complex formation of E-cadherin, catenins, and the phosphatidylinositol 3-kinase (PI3K), although the expression of these proteins was not affected. The assembly of the E-cadherin-catenin-PI3K complex was sensitive to the pharmacologic inhibition of Src family tyrosine kinases but was not affected by inhibition of Ca²⁺_o-induced rise in Ca²⁺_i. Inhibition of CaR expression blocked the Ca²⁺_o-induced tyrosine phosphorylation of β-, -, and p120-catenin, PI3K, and the tyrosine kinase Fyn and the association of Fyn with E-cadherin and PI3K. Our results indicate that the CaR regulates cell survival and Ca²⁺_o-induced differentiation in keratinocytes at least in part by activating the E-cadherin/PI3K pathway through a Src family tyrosine kinase-mediated signaling.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Keratinocytes proliferate and differentiate in cultures in a manner recapitulating the process of epidermal differentiation in vivo (1). Raising the [Ca²⁺] ²_o above 0.07 mM triggers an acute and then a sustained increase in Ca²⁺_i, and the onset of terminal differentiation (2, 3). Blocking the increase in Ca²⁺_i with BAPTA, a Ca²⁺_i chelator, inhibits the ability of Ca²⁺_o to induce differentiation (4, 5). Previous studies demonstrated an obligatory role for the CaR, a G-protein-coupled receptor, in mediating Ca²⁺ signaling and Ca²⁺_o-induced keratinocyte differentiation (6-8). Activation of the CaR by Ca²⁺_o stimulates phospholipase C (PLC) to convert phosphatidylinositol 4,5-bisphosphate into diacylglylcerol and inositol 1,4,5-trisphosphate. Inositol 1,4,5-trisphosphate in turn induces Ca²⁺ release from internal stores and increases Ca²⁺_i (9). Inhibition of CaR expression led to a decline of Ca²⁺_i pools and altered cellular Ca²⁺ handling, hence reducing the Ca²⁺_i response to Ca²⁺_o and impairing differentiation in keratinocytes (8).

One of the immediate cellular responses to Ca²⁺_o in epithelial cells is the formation of cell-cell contacts, a process mediated by the adhesion molecule E-cadherin (10). E-cadherin is a major classical cadherin in keratinocytes and is expressed throughout the epidermis (11). Upon Ca²⁺_o stimulation, the extracellular portion of E-cadherin interacts with E-cadherin molecules on the surface of neighboring cells, whereas its cytoplasmic tail interacts with β- (or -), -, and p120-catenins to form the core adhesive structure of adherens junctions (AJ) (12). E-cadherin-mediated cell adhesion plays key roles in remodeling of epithelial cell-cell interaction and maintaining proper epidermal differentiation (10, 13). Loss of E-cadherin in the epidermis leads to a loss of AJ and impaired terminal differentiation (14). The sequential binding of catenins physically links E-cadherin to the actin cytoskeleton and other signaling molecules, including phosphatidylinositol 3-kinase (PI3K) (10, 15). Keratinocyte differentiation induced by Ca²⁺_o necessitates the activation of PI3K. Pharmacological inhibition of PI3K blocks the expression of late differentiation markers and induces apoptosis in differentiating keratinocytes (16, 17). Through interactions with the E-cadherin-catenin complex PI3K is recruited to the cell membrane, where it converts phosphatidylinositol 4,5-bisphosphate to phosphatidylinositol 3,4,5-triphosphate. Phosphatidylinositol 3,4,5-triphosphate in turn binds and activates PLC1 (17), which is required for maintaining the Ca²⁺_o-induced increase in Ca²⁺_i crucial for keratinocyte differentiation (18). Inactivating E-cadherin function by specific antibody or blocking E-cadherin expression by small interfering RNA prevents Ca²⁺-induced activation of PI3K and, thus, keratinocyte differentiation (16, 19). Besides E-cadherin, our recent studies demonstrated that the Ca²⁺_o-induced recruitment of PI3K to cell membrane, activation of PI3K, and keratinocyte differentiation require β- and p120-, but not -catenin (19). Therefore, E-cadherin-dependent cell adhesion plays a key role not only in coordinating cellular organization and movement in epidermis, but also in transducing cellular signals that influence keratinocyte differentiation.

Keratinocyte differentiation induced by Ca²⁺_o is accompanied by increased tyrosine phosphorylation (17, 20, 21). Many studies have demonstrated that tyrosine kinase activity is necessary for the assembly of AJ and the interaction of PI3K with the E-cadherin-catenin complex. Pharmacological inhibition of tyrosine kinase perturbs the formation of AJ and prevents activation of PI3K by Ca²⁺_o (16, 22, 23). In differentiating mouse keratinocytes, Ca²⁺_o-induced assembly of E-cadherin-catenin complex and the recruitment of PI3K to E-cadherin are accompanied by tyrosine phosphorylation of β-, -, and p120-catenin (22). The stimulation of E-cadherin/PI3K signaling by Ca²⁺_o involves activation of Src family tyrosine kinases. Inhibiting Src family tyrosine kinases effectively blocks formation of AJ (22), abolishes Ca²⁺_o activation of PI3K, and suppresses expression of differentiation markers in keratinocytes (17). Consistent with the notion that Src family kinases control keratinocyte cell-cell adhesion, Fyn tyrosine kinase colocalizes with E-cadherin at the cell membrane (22). Additionally, Fyn-deficient keratinocytes exhibit decreased tyrosine phosphorylation of β-, -, and p120-catenin, abnormalities in cell adhesion (22), and compromised differentiation (21). Hence, in contrast to what has been found in transformed or mitogenically stimulated cells, tyrosine phosphorylation plays a positive role in control of cell adhesion in differentiating keratinocytes (24).

Ca²⁺_o activates CaR-mediated Ca²⁺_i signaling and E-cadherin-mediated cell-cell adhesion that lead to differentiation. In the present study, we investigated the involvement of CaR in the activation of E-cadherin signaling. Knocking down CaR expression blocked the Ca²⁺_o-induced formation of AJ, the association of PI3K with the E-cadherin-catenin complex, and expression of late differentiation markers. Furthermore, the Ca²⁺_o-induced tyrosine phosphorylation of β-, -, and p120-catenin and Fyn were blocked in the CaR-deficient keratinocytes. This indicates that the CaR regulates the E-cadherin/PI3K pathway via a Src family tyrosine kinase-mediated signaling and impacts on keratinocyte differentiation.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Materials—The membrane permeable Ca²⁺ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-AM (BAPTA-AM), 2-aminoethoxydiphenyl borate (2-APB), and Src kinase inhibitor PP2 and its non-functional analog PP3 were purchased from Calbiochem-Novabiochem Corp. (La Jolla, CA). Stock solutions of these compounds were prepared in Me₂SO. All other chemicals were purchased from Sigma. All DNA constructs used in generation of adenoviruses were prepared using Qiagen Maxi-prep columns (Chatsworth, CA) according to the manufacturer's protocol. Monoclonal antibodies (mAbs) for Bip (GRP78) and 2 integrin were obtained from BD Biosciences (Palo Alto, CA). The rabbit polyclonal antibody (Ab) for CaR, ADDR, was raised against the peptide corresponding to amino acids 215-236 of the human keratinocyte CaR (6). Rabbit polyclonal and mAbs against E-cadherin, -, β-, -, and p120-catenin, c-Src, Fyn, and the regulatory subunit of PI3K, p85, were from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Anti-phosphotyrosine mAb 4G10 was purchased from Upstate%20Biotechnology">Upstate Biotechnology Inc. (Lake Placid, NY). Texas Red-conjugated phalloidin was from Invitrogen Corp. (Carlsbad, CA).

Cell Culture—Normal human keratinocytes (NHKs) were isolated from neonatal human foreskins and grown in serum-free keratinocyte growth medium (154CF, Cascade Biologics, Portland, OR) as described (17). Briefly, keratinocytes were isolated from newborn human foreskins by trypsinization (0.25% trypsin, 4 °C, 16 h), and primary cultures were established in growth medium containing 0.07 mM CaCl₂. Second passage keratinocytes were plated in medium containing 0.03 mM CaCl₂ and used in the experiments described.

Generation of Adenoviral Stocks and Infection of Keratinocytes—A replication-defective adenovirus carrying the antisense human CaR cDNA (Ad-ASCaR) and the control viruses Ad-DNR were constructed using an Adeno-X Expression System II kit (BD Biosciences) as described (8). Viral particles were collected and titered using an Adeno-X rapid titer kit (BD Biosciences) and used to infect NHKs. For inactivation of the CaR, subconfluent NHKs were infected with the Ad-ASCaR adenovirus (60 pfu/cell) in growth medium containing 0.03 mM CaCl₂ and cultured for 5 to 7 days before exposure to 1.2 mM CaCl₂ for 5-10 min to induce formation of AJ or for 3 days to induce differentiation. Three days after infection, the viral supernatant was replaced with fresh culture medium containing 0.03 mM CaCl₂. No additional adenovirus was provided after the initial infection. Control cells were infected with an adenovirus Ad-DNR.

Quantitative Real-time PCR (q-PCR) Analysis—The expression of late differentiation genes was determined by q-PCR. NHKs were infected with adenovirus (60 pfu/cell) in growth medium containing 0.03 mM CaCl₂ and cultured for 5 days before exposure to 1.2 mM CaCl₂ for 3 days. Total RNA were then isolated using the Qiagen RNeasy RNA purification kit (Chatsworth, CA) according to the manufacturer's instructions. Equal amounts of RNA samples were reverse transcribed by Moloney murine leukemia virus reverse transcriptase (Invitrogen Crop.) to generate cDNA. qPCR was performed on cDNA using TaqMan premixed primer/probes and reagents from Applied Biosystems (Foster City, CA) or SYBR Green primers. RNA levels of late differentiation markers in keratinocytes were normalized to mitochondrial ribosomal protein L19 for all experiments.

TUNEL Staining—Keratinocyte cultures grown on glass coverslips were infected with adenovirus as described. Then cells were cultured in 0.03 or 1.2 mM CaCl₂ for 2 days before fixation in 10% neutral buffered formalin. Apoptotic cells were detected by ApopTag peroxidase in situ apoptosis detection kit (Chemicon International, Inc., Temecula, CA) according to the manufacturer's protocol. Briefly, fixed cells were washed and endogenous peroxidase activity is quenched by hydrogen peroxide. Cells were incubated with TdT enzyme in the presence of digoxigenin at 37 °C for 1 h, washed, and then incubated with peroxidase-conjugated anti-digoxigenin antibody. After washing with PBS, the sections are incubated with DAB substrate for 5 min to reveal peroxidase activity. Following the color reactions, the coverslips are washed and mounted. Digital images of 10 representative fields per experimental condition were acquired and quantified using a computer-assisted program (BIOQUANT, Nashville, TN). The degree of apoptosis was presented as the number of TUNEL-positive cells per 100 cells in the field. Student's t test was used for statistical analysis.

Measurement of Cytosolic Ca²⁺—The Ca²⁺_i responses to elevated Ca²⁺_i was measured using a Dual-wavelength Fluorescence Imaging System (Intracellular Imaging Inc., Cincinnati, OH) as described (25). Pre-confluent keratinocytes were infected with an adenovirus carrying the antisense human CaR cDNA (Ad-ASCaR) or a control virus (Ad-DNR) on a coverslip in medium containing 0.03 mM Ca²⁺. Five to 7 days later, cells were loaded with 5 µM Fura-2/AM (Molecular Probes, Eugene, OR) in 0.1% Pluronic F127 in buffer A (20 mM HEPES, 120 mM NaCl, 5 mM KCl, 1 mM MgCl₂, 1 mg/ml sodium pyruvate, 1 mg/ml glucose) containing 0.07 mM Ca²⁺. Cells were then washed and measured in buffer A containing 0.03 mM Ca²⁺ before exposure to 2 mM Ca²⁺. The cells were alternately illuminated with 340 and 380 nm light, and the fluorescence at emission wavelength 510 nm was recorded. All experiments were performed at room temperature. The signals from 20 to 50 single cells for each measurement were recorded. Each sample was calibrated by the addition of 20 µM ionomycin (R_max) followed by 20 mM EGTA/Tris, pH 8.3 (R_min). Cytosolic Ca²⁺ concentration ([Ca²⁺]_i) was calculated from the ratio of emission at the two excitation wavelengths based on the formula [Ca²⁺]_i = K_dQ(R - R_min)/(R_max - R), r = F₃₄₀/F₃₈₀, Q = F_min/F_max at 380 nm, and K_d for Fura-2 for Ca²⁺ is 224 nM.

Cell Lysate Preparation and Immunoblotting—Total cell lysates and membrane proteins were prepared from NHKs 5 to 7 days after adenoviral infections. Keratinocytes were washed twice in PBS containing 1 mM Na₃VO₄ and lysed for 30 min on ice in Nonidet P-40 lysis buffer (0.5% Nonidet P-40, 50 mM Tris-HCl, pH 8.0, 120 mM NaCl) supplemented with 1 mM phenylmethylsulfonyl fluoride and protease inhibitors (Complete^TM protease inhibitor tablet, Roche Molecular Biochemicals, Indianapolis, IN). Total cell lysates were centrifuged for 5 min at 4 °C and the supernatant was collected. Keratinocyte membrane lysates were prepared using the Mem-PER Eukaryotic Membrane Protein Extraction Reagent Kit (Pierce Biotechnology, Inc.) according to the manufacturer's instructions. The protein concentrations in the total cell lysates and membrane lysates were determined by the BCA Protein Assay Kit (Pierce Corp.). 50 µg of protein samples were electrophoresed through reducing polyacrylamide gels and electroblotted onto polyvinylidene fluoride membranes (Immobilon-P, 0.45 µm; Millipore Corp., Bedford, MA). After blocking with 5% milk in TBS (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA), the blots were incubated at 4 °C overnight with appropriate primary antibodies: polyclonal Abs against E-cadherin, - and p120-catenin at a dilution of 1:300, mAbs against PI3K-p85, phosphotyrosine, β- and -catenin at a dilution of 1:200, polyclonal Abs against Fyn and c-Src at a dilution of 1:200, mAbs against Bip and 2 integrin at a dilution of 1:250. Subsequently the blots were incubated with appropriate horseradish peroxidase-conjugated anti-IgG secondary antibodies (Amersham Biosciences, Piscataway, NJ) for 1 h at room temperature. The bound antibody was visualized using the SuperSignal West Dura Chemiluminescent Kit (Pierce Corp.) and subsequent exposure to x-ray film.

Immunoprecipitations—Total cell lysates containing 0.5-1 mg of protein were immunoprecipitated by 3 µg of mAbs against β-, -, and p120-catenin, Fyn, PI3K-p85, and phosphotyrosine (Santa Cruz Biotechnology, Inc.), and 200 µg membrane proteins by mAbs to E-cadherin and PI3K-p85 (Santa Cruz Biotechnology, Inc.), followed by Sepharose-conjugated protein G (ImmunoLink Immobilized Protein G, Pierce Corp.) in 1 ml of cold Nonidet P-40 lysis buffer at 4 °C with gentle tumbling overnight. Immunoprecipitates were washed 4 times in Nonidet P-40 lysis buffer, eluted, and then analyzed by Western analysis.

Immunofluorescence Staining—Keratinocytes were cultured on coverslips, fixed with 4% paraformaldehyde for 20 min at room temperature, and permeablized with 0.5% Nonidet P-40 in PBS for 5 min. After blocking with 5% goat serum in PBS, 0.01% Tween 20, cells were incubated with 10 µg/ml of primary antibodies at 4 °C for overnight. Subsequently cells were incubated with the appropriate fluorescein- or Texas Red-conjugated secondary antibody (20 µg/ml, Molecular Probes) at room temperature for 1 h. For F-actin staining, cells were incubated with Texas Red-conjugated phalloidin at room temperature for 1 h. Finally, coverslips were washed in PBS, mounted on glass slides using Gel-Mount (Biomeda, Foster City, CA) and examined with a Leica TCS NT/SP confocal microscope (Leica Microsystems, Heidelberg, Germany).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Inhibition of CaR Expression Reduced Ca²⁺_i Response to Ca²⁺_o, Promoted Apoptosis, and Suppressed Ca²⁺_o-induced Keratinocyte Differentiation—To study the role of CaR in activation of E-cadherin signaling and Ca²⁺_o-induced differentiation, we inhibited CaR expression by infecting preconfluent human keratinocytes with an adenovirus carrying a full-length CaR antisense cDNA (Ad-ASCaR). Immunoblotting (Fig. 1A) and immunostaining (Fig. 1B) showed that Ad-ASCaR effectively blocked the endogenous CaR protein level as compared with the cells infected with a control adenovirus (Ad-DNR). To determine whether inhibition of CaR expression altered Ca²⁺ signaling, we examined its impact on Ca²⁺_i response to Ca²⁺_o. Consistent with our previous studies (6, 8), knockdown of CaR inhibited the Ca²⁺_o-induced increase in Ca²⁺_i. As shown in Fig. 1C, raising Ca²⁺_o from 0.03 to 2 mM induced an increase in Ca²⁺_i in keratinocytes infected with the control virus Ad-DNR from 82 ± 7to472 ± 40 nM (mean ± S.D.; n = 49). Keratinocytes infected with the Ad-ASCaR virus had comparable resting Ca²⁺_i (71 ± 12 nM; n = 45), but had a marked reduction in the rise of Ca²⁺_i (to 202 ± 31 nM) in response to 2 mM Ca²⁺_o.As knockdown of CaR inhibited much of, but not completely blocked, the ability of Ca²⁺_o to increase Ca²⁺_i, other mechanisms, such as calcium channels, independent of CaR appear to play a role in mediating Ca²⁺_o-induced Ca²⁺_i response.

View larger version (31K): [in this window] [in a new window]   FIGURE 1. Inhibition of CaR expression by a CaR antisense cDNA blunted the acute Ca2+i response to elevated Ca2+o. Human keratinocytes were infected by an adenovirus carrying a full-length CaR antisense cDNA (Ad-ASCaR) or a control virus (Ad-DNR) at a titer of 60 plaque-forming units (pfu)/cells in medium containing 0.03 mM Ca2+. Five days after viral infection, endogenous CaR protein levels were assessed by: A, immunoblotting of total cell lysates and B, fluorescence immunostaining using a polyclonal antibody against CaR. Bar = 50 µm. Infection of the Ad-ASCaR effectively decreased the expression of endogenous CaR protein as compared with cells infected with Ad-DNR. After infection with Ad-ASCaR or Ad-DNR, keratinocytes were loaded with Fura-2. C, Ca2+i was measured before and after the addition of 2 mM Ca2+. The trace shown here represents the average Ca2+i of 45-49 individual keratinocytes during recording. The results are representative of three separate experiments.

View larger version (17K): [in this window] [in a new window]   FIGURE 2. Inhibition of CaR expression promoted apoptosis in keratinocytes. Keratinocytes were cultured on glass coverslips and infected with an Ad-ASCaR or a control (Ad-DNR) adenovirus as described. Then cells were grown in 0.03 or 1.2 mM CaCl2 for 2 days before being fixed for in situ TUNEL staining. Quantitative analysis of TUNEL-positive cells was performed as described under "Experimental Procedures." The level of apoptosis was expressed as number of TUNEL-positive cells per 100 cells in the field. The number of TUNEL-positive keratinocytes in the cultures infected with the Ad-ASCaR (black bars) was significantly higher than that in cultures infected with control Ad-DNR (white bars). *, p < 0.01. The data are representative of two independent experiments.

View larger version (25K): [in this window] [in a new window]   FIGURE 3. Inhibition of CaR expression blocked the Ca2+o-stimulated expression of late differentiation genes. Keratinocytes were infected with Ad-DNR or Ad-ASCaR adenoviruses for 5 days. Cells were either maintained in 0.03 mM or switched to 1.2 mM Ca2+ for 72 h, and total RNA was collected and analyzed for loricrin, filaggrin, caspase 14, and keratin 14 (K14) by real-time qPCR. Expressions of differentiation markers are presented as ratios to mitochondrial ribosomal protein L19. Raising Ca2+o from 0.03 to 1.2 mM significantly increased the mRNA levels of loricrin, filaggrin, and caspase 14 in keratinocytes infected with control virus (Ad-DNR, white bars). *, p < 0.01. The Ca2+-stimulated expression of late differentiation markers was blocked in cells infected with ad-ASCaR (black bars). The results are representative of three experiments.

To determine whether the decrease in CaR expression affected differentiation, we examined its impact on Ca²⁺_o-induced expression of terminal differentiation genes by quantitative real-time PCR in keratinocytes infected with Ad-ASCaR or Ad-DNR viruses. As shown in Fig. 3, 72 h of incubation in 1.2 mM Ca²⁺ significantly increased the mRNA levels of terminal differentiation markers loricrin, filaggrin, and caspase 14 in control keratinocytes infected with Ad-DNR (4-, 2.4-, and 5.5-fold increase, respectively), but failed to do so in the cells infected with Ad-ASCaR. However, neither Ca²⁺_o nor CaR knockdown decreased keratin 14 (K14), a marker for basal layer keratinocytes. These results demonstrated that CaR regulates calcium signaling, cell survival, and differentiation in keratinocytes.

View larger version (131K): [in this window] [in a new window]   FIGURE 4. Inhibition of CaR expression blocked the Ca2+o-induced assembly of intercellular adherens junctions. Keratinocytes were infected with Ad-DNR (A-D) or Ad-ASCaR (E-H) adenoviruses for 5 days. Cells were then exposed to 2 mM Ca2+ for 5 min to induce formation of cell-cell contacts. Keratinocytes were stained with a polyclonal antibody against E-cadherin, and monoclonal antibodies against β-(A and E), p120- (B and F), and -catenin (C and G), followed by the appropriate fluorescein isothiocyanate- or Texas Red-conjugated secondary antibody. F-actin was detected by Texas Red-conjugated phalloidin (D and H). Fluorescent signals were detected with a confocal microscope. Bars, A-C and E-G, 20 µm; D and H, 10 µm. Green (fluorescein isothiocyanate) and red (Texas Red) images were superimposed, so that sites of staining overlap are visualized as yellow. Substantial colocalization of E-cadherin with -, β-, and p120-catenin, as well as actin-cytoskeleton filaments was detected in the intercellular adherens junctions in control cells infected with Ad-DNR. The Ca2+o-induced formation of intercellular contacts was markedly inhibited in the keratinocytes infected with Ad-ASCaR. Similar observations were made in three separate cell preparations.

We then tested whether inhibition of CaR expression affects the production of the components of the E-cadherin signaling pathway. Immunoblotting analyses were performed on the total cell lysates from keratinocytes infected with Ad-ASCaR or Ad-DNR and treated with or without 2 mM Ca²⁺ using antibodies against E-cadherin, -, β-, -, and p120-catenins, and p85, the regulatory subunit of PI3K. Neither CaR knockdown nor the 5-min Ca²⁺ treatment significantly altered the expression levels of these proteins (data not shown). To confirm the immunolocalization results, we assessed whether CaR knockdown influences the cell membrane localization of these proteins. Immunoblotting analyses on the plasma membrane lysates demonstrated that 5 min of Ca²⁺ treatment promoted translocation of E-cadherin, -, β-, -, p120-catenin, and PI3K-p85 to the cell membrane in Ad-DNR-infected keratinocytes, whereas the Ca²⁺_o-induced membrane association of E-cadherin, catenins, and PI3K-p85 were markedly reduced in the Ad-ASCaR-infected cells (Fig. 5A). 2-Integrin was used as a control to demonstrate equal membrane protein extraction. The purity of the plasma membrane lysates was confirmed when only the antibody against the plasma membrane marker 2-integrin, but not the antibody against endoplasmic reticulum marker Bip, immunoreacted with the plasma membrane lysates.

We next carried out co-immunoprecipitation to examine whether CaR knockdown affects the complex formation of E-cadherin, catenins, and PI3K. Keratinocytes were infected with Ad-DNR or Ad-ASCaR then treated with 2 mM Ca²⁺ for 10 min. Plasma membrane lysates were incubated with a monoclonal antibody against either E-cadherin or PI3K-p85, and precipitated with protein G-conjugated Sepharose beads. The immunoprecipitates were then analyzed for the presence of E-cadherin, -, β-, -, p120-catenin, and PI3K-p85 by immunoblotting. As shown in Fig. 5B, Ca²⁺_o increased the complex formation of E-cadherin, -, β-, -, and p120-catenin, and PI3K-85 in Ad-DNR-infected keratinocytes, but it failed to do so in the Ad-ASCaR-infected cells. 2-Integrin was used as a control for equal membrane protein input for immunoprecipitation. Inhibition of CaR expression suppressed the complex formation of E-cadherin, catenins, and PI3K at the cell membrane, demonstrating a key role for CaR in the Ca²⁺_o activation of E-cadherin/PI3K pathway.

View larger version (32K): [in this window] [in a new window]   FIGURE 5. Inhibition of CaR expression blocked the Ca2+o-stimulated cell membrane localization and complex formation of E-cadherin, catenins, and PI3K. Keratinocytes were infected with Ad-DNR or Ad-ASCaR adenoviruses as described and then exposed to 2 mM Ca2+ for 5 min. A, the plasma membrane lysates were extracted, and analyzed by immunoblotting with antibodies against E-cadherin, -, β-, -, and p120-catenin, and p85 subunit of PI3K. B, the plasma membrane lysates were immunoprecipitated (IP) with antibody against either E-cadherin or PI3K-p85. The immunoprecipitates were then analyzed by immunoblotting for E-cadherin, -, β-, -, and p120-catenin, and PI3K-p85. 2-Integrin (a plasma membrane marker) was used as a control for equal extraction of cell membrane proteins. Ca2+ increased the levels and complex formations of E-cadherin, catenins, and PI3K-p85 in the plasma membrane in Ad-DNR-infected keratinocytes, but not in the Ad-ASCaR-infected cells. The data are representative of three experiments.

Studies of mouse keratinocytes have shown that Ca²⁺-induced tyrosine phosphorylation of β-, -, and p120-catenin is essential for their association with E-cadherin and the activation of PI3K, and that Fyn tyrosine kinase is likely involved in this process (22, 26). To investigate whether CaR regulates the Ca²⁺_o activation of E-cadherin-mediated cell-cell adhesion via tyrosine kinase-mediated signaling, we examined the impact of CaR knockdown on tyrosine phosphorylation of β-, -, and p120-catenin, and PI3K. Keratinocytes were infected with adenoviruses Ad-DNR or Ad-ASCaR, and exposed to 2 mM Ca²⁺ for 10 min to induce cell junction formation. Total cell lysates were prepared and immunoprecipitated with an antibody against phosphotyrosine. Immunoprecipitates were then analyzed by immunoblotting to detect β-, -, p120-catenin, PI3K-p85, and tyrosine kinases Src and Fyn. As shown in Fig. 7A, Ca²⁺_o increased the tyrosine phosphorylation of β-, -, p120-catenin, PI3K-p85, and Fyn, but not Src, in control keratinocytes, whereas the induction of tyrosine phosphorylation of these proteins by Ca²⁺_o was diminished in the Ad-ASCaR-infected cells. β-Actin was used as a control for equal protein input for immunoprecipitation. Similar findings were obtained when the total cell lysates were immunoprecipitated separately by antibodies against β-, -, p120-catenin, PI3K-p85, and Fyn, and immunoprecipitates were analyzed by immunoblotting for phosphotyrosine (Fig. 7B). Our results demonstrate that CaR controls the Ca²⁺_o-induced cell-cell adhesion via tyrosine kinase-mediated signaling.

To further determine the role of Src and Fyn in the Ca²⁺_o-induced cell-cell adhesion, we assessed whether these proteins associated with E-cadherin and PI3K at the cell membrane. Immunoblotting analyses on plasma membrane lysates of Ad-DNR- and Ad-ASCaR-infected keratinocytes demonstrated that Ca²⁺_o promoted the membrane localization of Fyn, not Src, to the cell membrane in control keratinocytes, but failed to do so in cells infected with Ad-ASCaR (Fig. 8A). Analyses of total cell lysates showed that the expression levels of Src and Fyn were not changed by Ca²⁺ exposure or CaR knockdown (Fig. 8B). Co-immunoprecipitation assays demonstrated that Ca²⁺_o induced recruitment of Fyn to the E-cadherin-PI3K complex at the cell membrane in control keratinocytes (Fig. 8C). But the Ca²⁺_o induction of the association of Fyn with E-cadherin and PI3K was blocked when CaR expression was inhibited by Ad-ASCaR (Fig. 8C). However, no association of Src with E-cadherin or PI3K in the cell membrane was detected by the co-immunoprecipitation assays (data not shown). These results indicate that CaR knockdown interferes with the Ca²⁺_o activation of E-cadherin-mediated cell adhesion due to reduced tyrosine kinase signaling that was likely mediated by the Fyn tyrosine kinase.

View larger version (63K): [in this window] [in a new window]   FIGURE 6. Src family tyrosine kinase-mediated signaling, but not Ca2+i, is required for the activation of E-cadherin-dependent intercellular adhesion by Ca2+o. Keratinocytes grown in 0.03 mM Ca2+ were pretreated for 30 min with 10 µM PP2, 25 µM BAPTA-AM, 75 µM 2-APB, or with 0.1% Me2SO (DMSO). A, keratinocytes were exposed to 2 mM Ca2+ for 5 min, stained with a polyclonal antibody against E-cadherin, and monoclonal antibody against β-catenin, followed by fluorescein isothiocyanate-conjugated anti-rabbit and Texas Red-conjugated anti-mouse antibodies. Bar = 20 µm. B, Ca2+i was measured before and after the addition of 2 mM Ca2+. The traces shown here represent the average Ca2+i of 31 to 55 individual cells during recording. C, keratinocytes were pretreated with 10 µM PP2, 10 µM PP3, or 0.1% Me2SO, exposed to 2 mM Ca2+ for 5 min. The plasma membrane lysates were extracted and immunoprecipitated (IP) withantibody against either E-cadherin or PI3K-p85. The immunoprecipitates were then analyzed by immunoblotting for E-cadherin and PI3K-p85. The E-cadherin-dependent intercellular adhesion and association of PI3K with E-cadherin was sensitive to the inhibition of Src family tyrosine kinases. These results are representative of two separate experiments.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

View larger version (34K): [in this window] [in a new window]   FIGURE 7. Inhibition of CaR expression diminished the Ca2+o-induced tyrosine phosphorylation ofβ-,-, and p120-catenin and PI3K. Keratinocytes were infected with Ad-DNR or Ad-ASCaR adenoviruses as described and then exposed to 2 mM Ca2+ for 10 min. A, total cell lysates were immunoprecipitated (IP) with antibody against phosphotyrosine (p-Tyr). The immunoprecipitates were analyzed by immunoblotting (IB) for β-, -, and p120-catenin, PI3K-p85, and tyrosine kinases Fyn and c-Src. β-Actin was used as a control for equal protein input before precipitation. B, in a reverse approach, total cell lysates were immunoprecipitated separately with antibodies against β-, -, and p120-catenin, PI3K-p85, and Fyn. The immunoprecipitates were analyzed by immunoblotting for phosphotyrosine. Ca2+ promoted the tyrosine phosphorylation of β-, - and p120-catenin, PI3K-p85, and Fyn in keratinocytes, but failed to do so when CaR expression was blocked by Ad-ASCaR. The data are representative of two experiments.

The CaR couples to multiple G proteins involved in distinct signaling pathways: G_i to inhibit the activity of adenylyl cyclase (31) and activate extracellular signal-regulated kinase (ERK) (32), G_q to stimulate PLC and phospholipase A2 (33), and Gβ to stimulate PI3K (34). In the present study, we placed Src family tyrosine kinase-mediated signaling downstream of CaR activation, as down-regulation of CaR in keratinocytes blocked the Ca²⁺_o-induced activation and association of Fyn with E-cadherin and PI3K, reduced the tyrosine phosphorylation of β-, -, and p120-catenins, and consequently inhibited cell adhesion and E-cadherin/PI3K signaling. However, the mechanism by which the CaR regulates Src family kinases is unclear. In mouse keratinocytes, Fyn is a downstream mediator of Rho A function in intercellular adhesion, because blocking endogenous Rho-GTPase activity inhibited Ca²⁺_o-induced Fyn activation, tyrosine phosphorylation of β-, -, and p120-catenins, and AJ formation (26). Several studies demonstrated the ability of CaR to activate Rho-mediated signaling by coupling to G_12/13 (35, 36) or G_q (37). CaR-mediated activation of Rho requires the physical interaction of the C-terminal region of CaR with the cytoskeletal protein filamin (38, 39). Filamin interacts with a pleckstrin homology domain of Rho guanine nucleotide exchange factor (Rho-GEF) (40) to link G and Rho A (41). This provides a possible mechanism by which the CaR regulates keratinocyte cell-cell adhesion.

Thus, Ca²⁺_o regulates cell survival and induces keratinocyte differentiation by at least two pathways. First, activation of CaR by Ca²⁺_o stimulates the PLC pathway to increase Ca²⁺_i and activate protein kinase C and downstream signaling events. Second, via a CaR-dependent mechanism Ca²⁺_o induces the formation of E-cadherin-mediated AJ, providing a scaffold for recruiting and activating other signaling molecules, such as PI3K, Akt, and PLC1, which is critical for differentiation. Disruption of CaR or either pathway results in abnormal differentiation in keratinocytes, although other mechanisms are likely also involved in regulating Ca²⁺_o-activated cellular responses.

View larger version (25K): [in this window] [in a new window]   FIGURE 8. Decreased expression of CaR blocked the Ca2+o-induced association of Fyn with E-cadherin and PI3K at the cell membrane. Keratinocytes were infected with Ad-DNR or Ad-ASCaR adenoviruses as described and then exposed to 2 mM Ca2+ for 10 min. The plasma membrane lysates (A) and total cell lysates (B) were extracted, and analyzed by immunoblotting with antibodies against Fyn and c-Src. 2-Integrin and Bip were used as controls for equal extraction of cell membrane and total lysate proteins, respectively. Whereas the total expression levels of Fyn and Src were not affected by Ca2+ or decreased CaR expression, Ca2+ selectively increased the level of Fyn in the plasma membrane in Ad-DNR-infected keratinocytes, but not in the Ad-ASCaR-infected cells. However, Ca2+ did not change the localization of Src. C, the plasma membrane lysates were immunoprecipitated (IP) with antibody against either E-cadherin or PI3K-p85. The immunoprecipitates were analyzed by immunoblotting for the presence of Fyn. Ca2+ promoted the association of Fyn with E-cadherin and PI3K in the cell membrane in Ad-DNR-infected keratinocytes, but not in the Ad-ASCaR-infected cells. The data are representative of two experiments.

	FOOTNOTES

¹ To whom correspondence should be addressed: Endocrine Unit (111N), Veterans Affairs Medical Center, 4150 Clement St., San Francisco, CA 94121. Tel.: 415-750-2089; Fax: 415-750-6929; E-mail: chia-ling.tu{at}ucsf.edu.

² The abbreviations used are: Ca²⁺_o, extracellular Ca²⁺;Ca²⁺_i, intracellular Ca²⁺; CaR, calcium sensing receptor; PLC, phospholipase C; AJ, adherens junction; PI3K, phosphatidylinositol 3-kinase; BAPTA, bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-AM; 2APB, 2-aminoethoxydiphenyl borate; mAb, monoclonal antibody; PBS, phosphate-buffered saline; NHK, normal human keratinocytes.

	ACKNOWLEDGMENTS

 
We thank Sally Pennypacker for technical help and the Cell Culture and Tissue Preparation Core of our program project for preparing human epidermal keratinocytes.

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TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 

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