The calcium-sensing receptor-dependent regulation of cell-cell adhesion and keratinocyte differentiation requires Rho and Filamin A

doi:10.1038/jid.2010.414

J Invest Dermatol. Author manuscript; available in PMC 2012 May 1.

Published in final edited form as:

J Invest Dermatol. 2011 May; 131(5): 1119–1128.

Published online 2011 January 6. doi: 10.1038/jid.2010.414

PMCID: PMC3078217

NIHMSID: NIHMS273045

The calcium-sensing receptor-dependent regulation of cell-cell adhesion and keratinocyte differentiation requires Rho and Filamin A

Chia-Ling Tu, Wenhan Chang, and Daniel D. Bikle

Endocrine Unit, Veteran Affairs Medical Center and University of California, San Francisco, California 94121

Address correspondence to Dr. Chia-Ling Tu, Endocrine Unit (111N), VA Medical Center, 4150 Clement Street, San Francisco, California 94121. Tel: (415) 7502089; Fax: (415) 7506929; Email: chia-ling.tu/at/ucsf.edu

The publisher's final edited version of this article is available free at J Invest Dermatol

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Abstract

Extracellular Ca²⁺ (Ca²⁺_o) acting through the calcium-sensing receptor (CaR) induces E-cadherin mediated cell-cell adhesion and cellular signals mediating cell differentiation in epidermal keratinocytes. Previous studies indicate that the CaR regulates cell-cell adhesion through the Fyn/Src tyrosine kinases. Here we investigate whether Rho GTPase is a part of the CaR-mediated signaling cascade regulating cell adhesion and differentiation. Suppressing endogenous Rho A expression by small interfering RNA (siRNA)-mediated gene silencing blocked the Ca²⁺_o-induced association of Fyn with E-cadherin, suppressed the Ca²⁺_o-induced tyrosine phosphorylation of β-, γ-, and p120-catenin and formation of intercellular adherens junctions. Rho A silencing also decreased the Ca²⁺_o-stimulated expression of terminal differentiation markers. Elevating Ca²⁺_o level induced interactions among CaR, Rho A, E-cadherin and a scaffolding protein filamin A at the cell membrane. Inactivation of CaR expression by adenoviral expression of a CaR antisense cDNA inhibited Ca²⁺_o-induced activation of endogenous Rho. Ca²⁺_o-activation of Rho required a direct interaction between the CaR and filamin A. Interference of CaR-filamin interaction inhibited Ca²⁺_o-induced Rho activation and the formation of cell-cell junctions. These results indicate that Rho is a downstream mediator of CaR in the regulation of Ca²⁺_o-induced E-cadherin mediated cell-cell adhesion and keratinocyte differentiation.

Introduction

Extracellular Ca²⁺ (Ca²⁺_o) is a critical regulator that promotes differentiation in epidermal keratinocytes. Raising the Ca²⁺_o concentration ([Ca²⁺]_o) above 0.1 mM induces an increase in intracellular free Ca²⁺ concentration ([Ca²⁺]_i) (Pillai and Bikle, 1991) and intercellular adhesion (Hennings and Holbrook, 1983). E-cadherin-mediated cell-cell adhesion plays a key role in maintaining the tissue integrity and differentiation of epidermal keratinocytes (Furukawa et al., 1997; Tinkle et al., 2004; Young et al., 2003). Raising [Ca²⁺]_o stimulates the binding of E-cadherin to its counterpart on the surface of neighboring cells, and its interactions with β- (or γ-), α-, and p120-catenins to form the core structure of adherens junctions (AJ) (Perez-Moreno et al., 2003; Pokutta and Weis, 2007). Through interactions with γ- and p120-catenin, phosphatidylinositol-3-kinase (PI3K) is recruited to the E-cadherin-catenin complex at the cell membrane (Calautti et al., 2005; Xie and Bikle, 2007) and in turn activates phospholipase C (PLC)-γ1 (Xie et al., 2005), which is required for maintaining the Ca²⁺_o-stimulated increase in Ca²⁺_i (Xie and Bikle, 1999) and keratinocyte differentiation (Xie and Bikle, 2007).

In keratinocytes, E-cadherin mediated cell-cell adhesion is regulated by the Src family tyrosine kinases, especially Fyn. Elevating [Ca²⁺]_o selectively activates Fyn kinase during differentiation and induces its association with the E-cadherin-catenin complex at the cell membrane (Calautti et al., 1998; Calautti et al., 2002). Intercellular adhesion and cell differentiation are compromised in Fyn-deficient keratinocytes (Calautti et al., 1998; Calautti et al., 1995). In addition to tyrosine kinases, the Rho family GTPases Rho and Rac are required for E-cadherin junction formation (Braga et al., 1997; Vaezi et al., 2002). Inhibiting Rho function by C3 toxin removes the E-cadherin complex from intercellular junctions (Braga, 1999; Braga et al., 1997), whereas expressing constitutively active Rho A promotes the AJ formation (Calautti et al., 2002). Perturbation of Rho A signaling also impedes terminal differentiation in keratinocytes (McMullan et al., 2003).

The Ca²⁺-sensing receptor (CaR) (Brown et al., 1993; Garrett et al., 1995), a member of family C of the G-protein coupled receptor (GPCR) superfamily, is expressed in the suprabasal cell layers in the epidermis (Komuves et al., 2002; Oda et al., 2000). It controls Ca²⁺ signaling (Oda et al., 1998; Tu et al., 2007) and the Ca²⁺_o-induced keratinocyte differentiation (Oda et al., 2000; Tu et al., 2001). Our recent studies indicate that the CaR regulates critical steps in E-cadherin-mediated cell-cell adhesion. Inhibiting CaR expression blocks the Ca²⁺_o-induced membrane translocation and activation of Fyn, the formation of the E-cadherin-catenin complex, activation of PI3K and, consequently, keratinocyte differentiation (Tu et al., 2008). How the CaR transduces Ca²⁺_o signals to intracellular responses in keratinocytes is unclear. In other cell systems, the CaR modulates multiple second messengers and intracellular signaling proteins, including inhibition of agonist-induced cAMP accumulation, Ca²⁺_o-stimulation of PLC and PLA2, and activation of mitogen-activated protein kinase (MAPK) (Huang et al., 2004; Rey et al., 2005). Evidence indicates that CaR-mediated signaling to MAPK necessitates the physical interaction between CaR and filamin A (Awata et al., 2001; Hjalm et al., 2001; Huang et al., 2006), a cytoskeletal actin-binding scaffolding protein that directly interacts with a variety of signaling proteins including GPCRs and Rho-like GTPases (Feng and Walsh, 2004; Stossel et al., 2001). Here we demonstrate that through forming a signaling complex with filamin and Rho the CaR elicits the Ca²⁺_o-activation of the E-cadherin-mediated pathway that impacts on keratinocyte differentiation.

Results

Inhibition of Rho A expression suppressed E-cadherin-mediated cell-cell adhesion and keratinocyte differentiation

To further delineate the role of Rho in E-cadherin-mediated cell-cell adhesion, we inhibited Rho A expression by transfecting human epidermal keratinocytes with a mixture of Rho A-specific siRNAs (siRhoA). Immunoblotting analyses showed that siRhoA effectively reduced the endogenous Rho A level as compared with the cells transfected with control siRNAs mix (siControl), whereas the expression of other similar small GTPases, Rac1 and cdc42, were not affected (Figure 1a). SiRNA-transfected keratinocytes were treated with 2 mM Ca²⁺ for 10 min to induce cell-cell adhesion. Fluorescence immunostaining for E-cadherin and β-catenin (supplementary Figure S1) revealed that the formation of intercellular adherens junctions was blocked in siRhoA-treated keratinocytes. Immunoblotting analyses of total cell extract and plasma membrane lysates (supplementary Figure S2) showed that while neither Ca²⁺ nor Rho A inhibition changed the expression levels of E-cadherin, β-, γ- and p120-catenin and Fyn (Figure S2a), the ability of Ca²⁺_o to increase membrane localization of these proteins of adherens junctions in control keratinocytes was blocked when Rho A expression was inhibited by siRhoA (Figure S2b). We then examined the levels of E-cadherin associated membrane cell-cell adhesion complex in these cells by co-immunoprecipitation. Plasma membrane lysates from siRNA-treated keratinocytes were precipitated with an antibody against either E-cadherin or Fyn, and protein G-conjugated sepharose beads. The immunoprecipitates were then analyzed for the presence of other components of the adhesion complex by immunoblotting. Ca²⁺_o increased the amount of E-cadherin-catenin-Fyn complex in the cell membrane of control keratinocytes, but this was markedly reduced in the siRhoA-transfected cells (Figure 1b), likely due to decreased membrane E-cadherin (see supplementary Figure S2b and S2e). In keratinocytes, Ca²⁺_o-induced tyrosine phosphorylation of β-, γ- and p120-catenin is essential to their association with E-cadherin and the stabilization of the adhesion complex, and this process is likely regulated by Fyn tyrosine kinase (Calautti et al., 1998; Calautti et al., 2002). We next examined the impact of Rho A knockdown on tyrosine phosphorylation of catenins in response to Ca²⁺_o. Total cell lysates were immunoprecipitated separately by antibodies against β-, γ-, p120-catenin and Fyn, and immunoprecipitates were analyzed for phosphotyrosine. As shown in Figure 1c, Ca²⁺_o- induced tyrosine phosphorylation of Fyn and β-, γ-, p120-catenin was diminished in the siRhoA-transfected keratinocytes as compared to control cells. Similar findings were obtained when total cell lysates were immunoprecipitated with an antibody against phosphotyrosine and immunoprecipitates analyzed for β-, γ-, p120-catenin, and Fyn (supplementary Figure S3). These observations indicate that Rho functions upstream of tyrosine kinase signaling in keratinocyte cell-cell adhesion.

Figure 1

Inhibiting Rho A expression by siRNA blocked cell-cell adhesion

To assess whether any functional defects associate with the biochemical changes of cell junction formation that result from Rho A inhibition, the cohesiveness of cell adhesion was evaluated by dispase dissociation assay (Figure 1d and supplementary Figure S4). Treatment of confluent keratinocytes cultured in 0.03 mM Ca²⁺ with dispase caused detachment of most cells (83 and 95% of control and siRhoA-treated keratinocytes, respectively) as single cell suspensions (Figure 1d). Although cultures switched to 2 mM Ca²⁺ displayed higher resistance to dispase, inhibiting Rho A expression resulted in a significant increase in dispase-released single cells (Figure 1d; siRhoA- vs. siControl-transfected keratinocytes 56 vs. 38% after 60-min calcium treatment and 33 vs. 15% after 150-min calcium treatment, P<0.05), indicative of a reduction in strength of cell adhesion.

Ca²⁺_o-stimulated keratinocyte differentiation is accompanied by a sustained increase in Ca²⁺_i (Sharpe et al., 1993). To investigate whether the perturbation of E-cadherin signaling following reduction in Rho A expression affects cell differentiation, we compared the Ca²⁺ _o-induced Ca²⁺_i accumulation and expression of the terminal differentiation genes in siRhoA-transfected keratinocytes with control cells. As shown in Figure 2a, raising [Ca²⁺]_o from 0.03 to 2 mM induced an increase in [Ca²⁺]_i in proliferating control keratinocytes from 110 ± 9 to a peak of 509 ± 28 nM (mean ± SD; n=43) with a plateau of 384 ± 14 nM. SiRhoA-transfected keratinocytes had comparable resting [Ca²⁺]_i (121 ± 11 nM; n=41); raising [Ca²⁺]_o from 0.03 to 2 mM elicited a transient surge in [Ca²⁺]_i (to 404 ± 31 nM), likely due to Ca²⁺ release from internal stores, but failed to maintain a sustained increase of Ca²⁺_i (Figure 2a). Inhibition of Rho A also blocked the ability of Ca²⁺_o to instigate a sustained increase in Ca²⁺_i in differentiating keratinocytes (Figure 2b). A 24-hr treatment with 1.2 mM Ca²⁺ significantly increased the basal [Ca²⁺]_i from 102 ± 11 to 144 ± 12 nM (n=33, P<0.05) in control keratinocytes, but failed to do so in siRNA-transfected cells (from 91 ± 5 to 82 ± 15nM; n=32). Next, we compared the expression of terminal differentiation genes after a 72-hr exposure to 1.2 mM Ca²⁺in siRNA-transfected keratinocytes by immunoblotting. As shown in Figure 2c, Ca²⁺_o-induced expression of the terminal differentiation marker proteins keratin 1 (K1), involucrin, transglutaminase (TG) 1 and loricrin were considerably decreased in siRhoA-treated keratinocytes as compared to control cells, whereas neither Ca²⁺ nor Rho A knockdown altered the protein level of the basal keratinocyte marker keratin 14 (K14).

Figure 2

Inhibiting Rho A expression suppressed Ca²⁺_o-induced increase of [Ca²⁺]_i and keratinocyte differentiation

Rho A is a downstream mediator in CaR-dependent regulation of E-cadherin mediated cell-cell adhesion

Our recent studies indicate that inhibiting the expression of CaR in keratinocytes blocks the ability of Ca²⁺_o to induce the E-cadherin-mediated cell-cell adhesion (Tu et al., 2008). Endogenous CaR expression was inhibited by infecting keratinocytes with an adenovirus carrying a full-length CaR antisense cDNA (Ad-ASCaR). Immunoblotting (Figure 3a) showed that Ad-ASCaR effectively diminished the CaR protein level as compared with the cells infected with a control adenovirus (Ad-DNR). To determine whether Rho is a part of the CaR-mediated signaling cascade, we examined the impact of CaR knockdown on endogenous Rho activity in response to Ca²⁺_o. Keratinocytes infected with Ad-ASCaR or Ad-DNR were exposed to 2 mM Ca²⁺ for 10 min. Total cell lysates were analyzed by pull-down assays with the Rho-binding domain (RBD) of rhotekin, which specifically binds the GTP-bound activated form of Rho (GTP-Rho). While neither Ca²⁺_o nor CaR knockdown affected total Rho levels, the ability of Ca²⁺_o to increase endogenous Rho activity in control keratinocytes was blocked when CaR was knocked down by Ad-ASCaR (Figure 3b). Immunoblotting of the plasma membrane lysates (supplementary Figure S5) demonstrated that Ca²⁺_o promoted translocation of Rho, predominantly the active form, to the cell membrane in control keratinocytes, but failed to do so in the Ad-ASCaR-infected cells, signifying the importance of the CaR in Ca²⁺_o-activation of Rho.

Figure 3

Inhibition of CaR expression by a CaR antisense cDNA abolished the Ca²⁺_o-induced activation of Rho

To determine whether the CaR activates Rho signaling through direct protein interactions, we immunoprecipitated keratinocyte plasma membrane lysates using an antibody against either E-cadherin or CaR and followed by protein G-conjugated sepharose beads. Immunoprecipitates were then analyzed by immunoblotting for the presence of CaR, E-cadherin, Rho A and filamin A. Ca²⁺_o promoted the association of CaR, Rho A, E-cadherin and filamin A at the cell membrane (Figure 4a). Fluorescence immunostaining showed that CaR (Figure 4b), Rho A (Figure 4c) and filamin A (Figure 4d) translocated with E-cadherin from cytosol to cell-cell junctions in response to elevated [Ca²⁺]_o. These observations suggested that CaR elicited the Ca²⁺_o-activation of E-cadherin-medicated pathways by forming a signaling complex with Rho A, filamin and E-cadherin at the cell-cell junctions.

Figure 4

Elevated [Ca²⁺]_o induced complex formation of CaR, , Rho A, filamin A and E-cadherin at the cell membrane

Ca²⁺_o-activation of Rho A and E-cadherin-mediated cell-cell adhesion required a physical interaction between CaR and filamin

Several studies have determined that the mid-portion of the CaR Carboxyl-terminus (amino acids 906–980) directly interacts with the region of filamin A that contains repeats 14–17 and hinge 1 (amino acids 1530–1875) (Awata et al., 2001; Hjalm et al., 2001; Zhang and Breitwieser, 2005). In order to determine whether CaR-mediated regulation of cell-cell adhesion in keratinocytes requires filamin, we blocked the endogenous CaR-filamin interaction by infecting keratinocytes with adenoviruses that express either a Myc-tagged CaR C-terminus peptide (Ad-CaRL, amino acid 907–980) or a HA-tagged filamin A peptide containing domains 15–16 (Ad-FLNaL, amino acid 1566–1875), then examined their impact on AJ formation. Immunoprecipitation of Ad-CaRL-infected keratinocyte lysates using an anti-Myc antibody brought down filamin A, whereas an anti-HA antibody pulled down CaR in Ad-FLNaL-infected cells, confirming direct interactions of these peptides with their respective native binding partners (Figure 5a). To test whether expressing these peptides affects Ca²⁺_o-induced Rho activation, Ad-DNR, Ad-CaRL- and Ad-FLNaL-infected keratinocytes were exposed to 2 mM Ca²⁺ for 10 min, and Rho activity in total cell extracts (supplementary Figure S7) and plasma membrane lysates (Figure 5b) was determined based on the level of GTP-bound Rho (Rho-GTP). Although neither Ca²⁺ nor expressing dominant-negative CaR or filamin peptides changed the expression level of Rho A (Figure S7), interference of the CaR-filamin interaction by Ad-CaRL or Ad-FLNaL inhibited the ability of Ca²⁺_o to promote cell membrane localization (Figure 5b) and endogenous Rho A activity (Figure 5b and Figure S7). To assess whether the interaction between endogenous CaR and filamin was disrupted by these peptides, we immunoprecipitated plasma membrane lysates using an antibody against CaR. Immunoblotting analyses of immunoprecipitates for filamin A showed that Ca²⁺_o promoted the association of CaR and filamin A in the cell membrane in control keratinocytes but not in cells infected with either Ad-CaRL or Ad-FLNaL (Figure 5c). Consistent with the finding that the interaction with filamin is essential for the surface expression of CaR (Zhang and Breitwieser, 2005), the ability of Ca²⁺_o to increase the protein level of CaR in the plasma membrane was diminished in the Ad-CaRL- and Ad-FLNaL-infected keratinocytes (Figure 5c). Interfering with the CaR-filamin interaction by Ad-CaRL- and Ad-FLNaL also blocked the Ca²⁺_o-promoted complex formation of CaR, Rho A, E-cadherin and filamin in the cell membrane (Figure 5c), likely due to diminished membrane CaR and Rho A. Similar results were obtained when the plasma membrane lysates were immunoprecipitated with an antibody against E-cadherin (Figure 5d).

Figure 5

Disrupting the CaR-filamin interaction blocked the Ca²⁺_o-induced Rho activation and membrane complex formation of CaR, Rho A and E-cadherin

Fluorescence immunostaining showed that Ad-CaRL or Ad-FLNaL inhibited the ability of Ca²⁺_o to induce colocalization of E-cadherin with β-catenin (Figure 6a), Rho A (Figure 6b) and filamin A (Figure 6c) at the cell-cell junctions. Dispase treatment showed that interfering with the CaR-filamin interaction markedly increased the susceptibility of intercellular junctions to dispase (Figure 6d and supplementary Figure S8). After 60 min of calcium treatment only 37% of control keratinocytes were released into suspension as single cells as opposed to 75% of Ad-CaRL- and 57% of Ad-FLNaL-infected cells (Figure 6d). Although keratinocytes switched to 2 mM Ca²⁺ for 150 min had formed stronger adherens junctions, Ad-CaRL and Ad-FLNaL caused a 50% reduction in strength of cell adhesion (Figure 6d). These results indicate that the Ca²⁺_o-induced E-cadherin-mediated cell-cell adhesion was reduced by blocking the CaR-filamin interaction due to inhibited Rho signaling.

Figure 6

Interfering with the CaR-filamin interaction suppressed Ca²⁺_o-induced formation of adherens junctions

Discussion

Ca²⁺_o induces keratinocyte differentiation by at least two pathways: First, Ca²⁺_o increases Ca²⁺_i and activates protein kinase C and downstream signaling events through stimulating the PLC pathway. Second, Ca²⁺_o induces the assembly of E-cadherin-mediated intercellular junctions, providing a framework for engaging and activating other signaling molecules, such as PI3K, Akt and PLCγ1, that is important for differentiation. We previously demonstrated that CaR controls the Ca²⁺_o-activation of Ca²⁺_i, E-cadherin-mediated signaling and keratinocyte differentiation (Tu et al., 2007; Tu et al., 2008). The inhibition of E-cadherin-mediated cell-cell adhesion in CaR-deficient keratinocytes is due to an ineffectual upstream Rho- and Src-family tyrosine kinase-dependent signaling. Rho is an upstream mediator of tyrosine kinase signaling in intercellular adhesion, since inhibiting endogenous Rho expression by siRNA blocks the Ca²⁺_o-induced membrane translocation and activation of Fyn, the tyrosine phosphorylation of β-, γ-, and p120-catenins and the formation of the E-cadherin-catenin adhesion complex. In the present study, we placed Rho-mediated signaling downstream of CaR activation, as CaR knockdown prevented Ca²⁺_o-activation of Rho and its association with E-cadherin at cell-cell junctions. Though keratinocyte intercellular adhesion is independent of the level of Ca²⁺_i (Tu et al., 2008), the E-cadherin-dependent signaling cascade provides a mechanism for maintaining an elevated Ca²⁺_i level in response to Ca²⁺_o, which is vital for keratinocyte differentiation (Xie and Bikle, 2007; Xie et al., 2005). Inhibition of E-cadherin signaling following Rho A knockdown crippled the ability of Ca²⁺_o to sustain an increase in Ca²⁺_i, although a transient increase of [Ca²⁺]_i still occurred; hence, the expression of differentiation marker genes was suppressed in the Rho A-deficient keratinocytes. These results indicate that CaR regulates both cell-cell adhesion and keratinocyte differentiation at least partly through the Rho/Fyn-mediated signaling pathway.

How CaR transduces Ca²⁺_o signals to activate downstream Rho pathway in keratinocytes is unclear. Here we demonstrated that Ca²⁺_o-activation of Rho requires the physical interaction of the C-terminal region of CaR with filamin A. Filamin A is known to interact with intracellular signaling proteins including the Rho-like GTPase, Rho guanine nucleotide exchange factor, Rho kinase, MAPK, SMADs and phosphatases (Feng and Walsh, 2004; Stossel et al., 2001). Filamin A also directly interacts with a variety of transmembrane proteins including β integrins, Ca and K channels, the insulin receptor and a subset of GPCRs (dopamine D2 receptor, metabotropic glutamate receptor, μ-opioid receptor, the calcitonin receptor and CaR) (Feng and Walsh, 2004; Stossel et al., 2001). In HEK293 and M2 cells, raising [Ca²⁺]_o enhances interactions between the CaR and filamin A. This interaction with filamin A increases plasma membrane localization of CaR, facilitating CaR signaling to the MAPK pathway (Zhang and Breitwieser, 2005). Disturbing the CaR-filamin interaction blocks CaR-mediated activation of extracellular signal-regulated kinase (Awata et al., 2001; Hjalm et al., 2001). In keratinocytes, raising [Ca²⁺]_o induces interactions among the CaR, filamin A, Rho A and E-cadherin at the cell membrane, suggesting that CaR mediates the Ca²⁺_o-activation of E-cadherin-mediated pathways by forming a signaling complex with Rho A via filamin. Consistent with the notion that filamin controls cell organization and trafficking of its interacting proteins (Liu et al., 1997; Seck et al., 2003; Zhang and Breitwieser, 2005), disrupting CaR-filamin interaction in keratinocytes by expressing a dominant-negative filamin A or a CaR C-terminus peptide inhibited the Ca²⁺_o-promoted plasma membrane expression of CaR and membrane translocation and activation of Rho and, consequently, E-cadherin-mediated cell-cell adhesion.

Altogether, our results show that Rho A is a downstream mediator of CaR and upstream of tyrosine kinase-mediated signaling in regulating cell-cell adhesion and differentiation in keratinocytes.

Materials and Methods

Cells and antibodies

Human neonatal foreskin keratinocytes (NHK) were cultured in serum-free growth medium (154CF, Cascade Biologics, Portland, OR) containing 0.03 mM CaCl₂ and used in the experiments described. The following antibodies (Abs) were used in this study: monoclonal antibodies (mAbs) for α2 integrin (BD Biosciences, San Jose, CA), for human involucrin and β-actin, for c-Myc- and HA-tags (Sigma-Aldrich, St. Louis, MO), polyclonal and monoclonal Abs against E-cadherin, β-, γ-, and p120-catenin and Fyn, polyclonal Abs against Rho A, Rac1 and cdc42, and CaR, mAb against transglutaminase 1 (Santa Cruz Biotechnology, Santa Cruz, CA), mAbs for phosphotyrosine, Rho and filamin A (Millipore Corp., Temecula, CA), polyclonal Abs for human keratin 1, keratin 14 and loricrin (Covance Inc., Berkeley, CA), and rabbit anti-human CaR (Tu et al., 2001). The study was conducted according to the Declaration of Helsinki Principles, and the use of human keratinocytes was approved by the committee for Human Research at the University of California San Francisco and the San Francisco Veteran Affair Medical Center.

SiRNA-mediated gene silencing

SiRNAs (siGENOME SMART pool reagent) for human Rho A (siRhoA) and negative control (siControl) were purchased from Dharmacon Inc. (Lafayette, CO). Pre-confluent NHKs were transfected with 100 nM siRNA using TransIT-siQUEST transfection reagent (Mirus BioLLC, Madison, WI). Three days later cells were exposed to 2 mM CaCl₂ for 10 minutes to induce formation of cell-cell contacts or to 1.2 mM CaCl₂ for 72 hours to induce differentiation.

Generation of adenoviral stocks and infection of keratinocytes

The cDNAs encoding the CaR C-terminal tail and the filamin A domains 15–16 were amplified from human keratinocytes by PCR using specific primers (see supplementary Figure S6). Replication-defective adenoviruses carrying the cDNAs for full-length antisense human CaR (Ad-ASCaR) (Tu et al., 2007), the CaR C-terminal filamin-interacting domain (Ad-CaRL), CaR-interacting region of filamin A (Ad-FLNaL), and the control adenovirus (Ad-DNR) were constructed using an Adeno-X Expression System II kit (BD Biosciences) and produced in HEK293 cells. Sub-confluent NHKs were infected with adenoviruses at a titer of 60–100 pfu/cell in growth medium containing 0.03 mM CaCl₂ and cultured for 5 days before exposure to 2 mM CaCl₂ for 10 minutes to induce cell-cell contacts.

Dispase dissociation of keratinocyte

Triplicate wells of confluent keratinocytes cultures in medium containing either 0.03 or 2 mM CaCl₂ were washed in PBS and incubated in 2.5 U/ml dispase solution (BD Biosciences) at 37°C for 15 min. Cells from each sample, including single cells released into media and detached cell sheets, were collected by scraping, and then washed with PBS. After centrifugation keratinocytes were resuspended in PBS by pipetting 15 times with 1 ml pipet tips. The number of single cells in suspension was counted using a hemocytometer. The whole samples were then centrifuged and incubated with 0.05% trypsin at 37°C for 10 min to release all cells into suspension. Total cells counts were used for normalization.

Measurement of cytosolic Ca²⁺

The cytosolic Ca²⁺ concentration ([Ca²⁺]_i) in keratinocytes in response to elevated [Ca²⁺]_o was measured using a Dual-wavelength Fluorescence Imaging System (Intracellular Imaging Inc., Cincinnati, OH) as described (Tu et al., 2001). To measure the acute Ca²⁺_i responses to elevated Ca²⁺_o, siRNA-transfected NHKs on coverslips were loaded with 5 μM Fura-2/AM (Molecular Probes, Eugene, OR) and maintained in 0.03 mM CaCl₂. [Ca²⁺]_i was measured before and after exposure to 2 mM CaCl₂. Also, keratinocyte cultures were maintained in growth medium containing 0.03 or 1.2 mM CaCl₂ for 24 hrs before [Ca²⁺]_i measurement. The data shown represent the average [Ca²⁺]_i of 32–43 individual keratinocytes during recording.

Cell lysate preparation, immunoblotting and immunoprecipitations

Conditions for preparing total keratinocyte lysates and membrane proteins, immunoblotting and immunoprecipitation were as described previously (Tu et al., 2008). 50 μg protein samples were used in immunoblotting analyses. Total cell lysate containing 1 mg protein or 200 μg membrane protein were immunoprecipitated by 3 μg of designated antibodies, followed by Sepharose-conjugated protein G (Pierce Corp.).

Fluorescence immunostaining

Keratinocytes were cultured on coverslips, fixed with 4% paraformaldehyde, and permeablized with 0.5% NP-40 in PBS. Conditions for staining were as described previously (Tu et al., 2008). After incubation with 10 μg/ml of primary antibodies and, subsequently, with 10 μg/ml of the appropriate fluorescein- or Texas red-conjugated secondary antibody (Invitrogen Corp.), coverslips were mounted and examined with a Leica TCS NT/SP confocal microscope (Leica Microsystems, Heidelberg, Germany).

Rho activity

Adenovirus-infected keratinocytes were exposed to 2 mM Ca²⁺ for 10 minutes and then lysed in MLB buffer (1% NP-40, 25 mM Tris-HCl, pH 7.4, 150 mM NaCl, 10 mM MgCl₂, 1 mM EDTA, 1 mM Na₃VO₄, 20 mM NaF) supplemented with protease inhibitors and phosphatase inhibitors. Total cell extracts or plasma membrane lysates were incubated with 20 μg of agarose-conjugated GST-rhotekin Rho binding domain fusion protein (Millipore Corp.), which binds specifically to GTP-bound Rho, in cold MLB buffer at 4°C overnight. Precipitates were washed, eluted, and then evaluated by immunoblotting analysis for Rho.

Statistical analysis

Student’s t-Test (for comparing two sample groups) and one-way ANOVA (for comparing more than two groups) were performed to analyze the quantitated data of various assays described in this study. Statistical difference is determined significant if P value is lower than 0.05.

Supplementary Material

Supplementary figures

Click here to view.^{(4.5M, pdf)}

Acknowledgments

We thank Sally Pennypacker and the Cell Culture and Tissue Preparation Core for providing human epidermal keratinocytes. This work was supported by a Merit Review Award from the Department of Veterans Affairs (to D.B) and grants PO1-AR39448, R21-AR38386, RO1-AG21353 and RO1-AR056256 from the National Institutes of Health.

Abbreviations


Ca²⁺_o	extracellular Ca²⁺

Ca²⁺_i	intracellular Ca²⁺

CaR	calcium-sensing receptor

PLC	phospholipase C

AJ	adherens junction

PI3K	phosphatidylinositol 3-kinase

siRNA	small interfering RNA

GPCR	G-protein coupled receptor

MAPK	mitogen-activated protein kinase

Footnotes

Conflict of Interest

The authors state no conflict of interest.

Supplementary Material

Supplementary material is linked to the online version of the paper at http://www.nature.com/jid

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