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Vol. 17, Issue 10, 4446-4458, October 2006

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Up-Regulation of Transient Receptor Potential Canonical 1 (TRPC1) following Sarco(endo)plasmic Reticulum Ca²⁺ ATPase 2 Gene Silencing Promotes Cell Survival: A Potential Role for TRPC1 in Darier's Disease

Biswaranjan Pani^*, Eric Cornatzer^*, William Cornatzer, Dong-Min Shin, Mark R. Pittelkow, Alain Hovnanian^||, Indu S. Ambudkar^¶, and Brij B. Singh^*

Departments of *Biochemistry and Molecular Biology and Internal Medicine, School of Medicine and Health Sciences, University of North Dakota, Grand Forks, ND 58202; Department of Oral Biology, Korea 21 Project for Medical Science, Yonsei University College of Dentistry, Seoul 120-752, Korea; Department of Dermatology, Mayo Clinic College of Medicine, Rochester, MN 55905; ^||Department of Functional Genetics of Epithelial Diseases, Institut National de la Santé et de la Recherche Médicale U563, 31024 Toulouse Cedex 3, France; and ^¶Secretory Physiology Section, Gene Therapy and Therapeutics Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD 20892

Submitted March 30, 2006; Revised July 18, 2006; Accepted August 2, 2006
Monitoring Editor: M. Bishr Omary

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The mechanism(s) involved in regulation of store operated calcium entry in Darier's disease (DD) is not known. We investigated the distribution and function of transient receptor potential canonical (TRPC) in epidermal skin cells. DD patients demonstrated up-regulation of TRPC1, but not TRPC3, in the squamous layers. Ca²⁺ influx was significantly higher in keratinocytes obtained from DD patients and showed enhanced proliferation compared with normal keratinocytes. Similar up-regulation of TRPC1 was also detected in epidermal layers of SERCA2^+/– mice. HaCaT cells expressed TRPC1 in the plasma membrane. Expression of sarco(endo)plasmic reticulum Ca²⁺ ATPase (SERCA)2 small interfering RNA (siRNA) in HaCaT cells increased TRPC1 levels and thapsigargin-stimulated Ca²⁺ influx, which was blocked by store-operated calcium entry inhibitors. Thapsigargin-stimulated intracellular Ca²⁺ release was decreased in DD cells. DD keratinocytes exhibited increased cell survival upon thapsigargin treatment. Alternatively, overexpression of TRPC1 or SERCA2-siRNA in HaCaT cells demonstrated resistance to thapsigargin-induced apoptosis. These effects were dependent on external Ca²⁺ and activation of nuclear factor-B. Isotretinoin reduced Ca²⁺ entry in HaCaT cells and decreased survival of HaCaT and DD keratinocytes. These findings put forward a novel consequence of compromised SERCA2 function in DD wherein up-regulation of TRPC1 augments cell proliferation and restrict apoptosis. We suggest that the anti-apoptotic effect of TRPC1 could potentially contribute to abnormal keratosis in DD.

	INTRODUCTION

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Darier's disease (DD) is an autosomal dominant inherited skin disease, which is characterized by the loss of adhesion between epidermal cells and abnormal keratinization (Burge and Wilkinson 1992). Typical histological findings include focal areas of separation between suprabasal epidermal cells, unusual dyskeratosis with round dyskeratotic keratinocytes (Munro, 1992). Mutations in the sarco(endo)plasmic reticulum Ca²⁺ ATPase (SERCA) isoform 2b (SERCA2b) gene leads to a loss of function, and these mutations have been shown to cause DD (Sakuntabhai et al., 1999). Most physiological systems are able to compensate for the loss of function of SERCA2b, possibly because of expression of other SERCA isoforms within those tissues (Dhitavat et al., 2003; Tavadia et al., 2004). However, of the multiple isoforms of SERCA1, -2, or -3, only SERCA2b is expressed in keratinocytes (Lytton and MacLennan, 1988; Ruiz-Perez et al., 1999). The mutated SERCA2 fails to sequester cytosolic Ca²⁺ into the endoplasmic reticulum (ER) lumen, thereby disturbing the otherwise normal Ca²⁺ homeostasis circuitry within the cells (Zhao et al., 2001; Ahn et al., 2003). Although oral retinoids, such as isotretinoin, have been shown to reduce hyperkeratosis (Burge and Wilkinson, 1992), the mechanism behind dysfunction of SERCA2 resulting in keratosis remains elusive.

Ca²⁺ is an integral signaling element that regulates numerous cellular processes (Clapham, 1995; Berridge et al., 2000). In skin cells, Ca²⁺ is potentially a vital component for differentiation, proliferation, and the formation of epithelial cell junctions (Tavadia et al., 2001). The action of Ca²⁺ ion in cells is dependent on its cellular concentration as well as its compartmentalization (Berridge et al., 2000). Ca²⁺ exerts a biphasic effect on cellular growth, a modest increase in cytosolic [Ca²⁺]_i can promote cell proliferation, whereas relatively high cytosolic [Ca²⁺] can lead to increased mitochondrial [Ca²⁺]_i and account for the release of proapoptotic proteins resulting in cell death (Anglade et al., 1997; Tompkins et al., 1997). Cytosolic [Ca²⁺] depends on release from internal stores, entry from external medium as well as removal of Ca²⁺ via the action of plasma membrane Ca²⁺ ATPase and SERCA pumps (Minke and Cook, 2002; Putney, 2003; Montell, 2005). There is strong evidence that release of Ca²⁺ from the intracellular stores is initiated by activation of the G protein-coupled receptor that leads to phosphatidylinositol bisphosphate (PIP₂) breakdown and generation of the second messenger inositol-1,4,5-trisphosphate (IP₃). IP₃ subsequently binds to its receptor in the ER and induces release of Ca²⁺ from internal stores. Depletion of the intracellular stores also leads to the opening of the plasma membrane Ca²⁺ channels, referred to as store-operated channel via an as of yet unknown mechanism (Putney, 2004). Treatment of cells with reagents that cause inhibition of intracellular Ca²⁺ accumulation, such as thapsigargin (Tg; a SERCA pump blocker) initiates a passive release of Ca²⁺ from the internal stores (Putney, 2003) and activates the same Ca²⁺ entry pathway. Interestingly, treatment of cells with Tg also leads to apoptosis and cell cycle arrest. Other receptor-dependent channels that are not linked to internal Ca²⁺ store depletion are also activated by agonist-stimulated PIP₂ hydrolysis.

Mammalian homologues of the Drosophila transient receptor potential (trp) gene have been suggested as components of plasma membrane Ca²⁺ influx channels (Putney, 2003; Venkatachalam et al., 2003; Montell, 2005). In recent years, 28 mammalian members of the TRP superfamily have been identified, which are activated by diverse stimuli including heat, pressure, activation of G protein-coupled receptors, and intracellular store depletion (Moran et al., 2004). TRP channels are involved in a multitude of physiological events, which include secretion, pain transduction, osmoregulation, mechanosensitivity, cell growth, and differentiation (Moran et al., 2004; Pla et al., 2005).

Growth and differentiation of keratinocytes is exquisitely determined by the [Ca²⁺]_i in the medium and the cytosol as well. However, the mechanism of this Ca²⁺-dependent regulation is not elaborately investigated. Our previous studies demonstrated that transient receptor potential canonical (TRPC)1 functions as store-operated calcium entry (SOCE) channel (Liu et al., 2000; Singh et al., 2001). Furthermore, a role of TRPC1 in the proliferation of neuronal and epithelial cells has been shown (Bollimuntha et al., 2005). Because SERCA2 are key molecules regulating store-operated calcium entry, the present study was undertaken to determine the role of TRPC channels in SERCA-compromised skin cells. Our data demonstrate that epidermal expression of TRPC1 is significantly increased in skin samples obtained from DD patients and SERCA^+/– mice. An increase in Ca²⁺ influx was also seen in DD keratinocytes. As an alternative approach to SERCA2 dysfunction in DD, expression of SERCA2-small interfering RNA (siRNA) in a human epidermal keratinocyte (HaCaT) cell line also exhibited an increase in TRPC1 expression and Ca²⁺ entry as well. This Ca²⁺ influx was inhibited by isotretinoin and SOCE blockers. Importantly, HaCaT cells overexpressing TRPC1 showed higher levels of the antiapoptotic protein Bcl-xL_, whereas no change in the expression of caspase-8, -9, and Apaf1 proteins was observed. Also, upon stimulation with Tg, cells overexpressing TRPC1 or SERCA-siRNA showed an enhanced activation of nuclear factor-B (NF-B) and resistance to apoptosis. This suggests overexpression of TRPC1 confers to an apoptotic-resistant phenotype in keratinocytes, thereby augmenting net cell survival in Tg-induced apoptosis. Together our data suggest a novel role for TRPC1 in the survival of keratinocytes with SERCA2 dysfunction. Thus, TRPC1 could contribute to the skin cell pathologies associated with DD.

	MATERIALS AND METHODS

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HaCaT, Primary and DD Keratinocytes Culture, Transformation, and Reagents
HaCaT cells were cultured in DMEM (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum, 1 U/ml penicillin, and 1 µg/ml streptomycin, and they were maintained at 37°C with 95% humidified air and 5% CO₂ as described in Boukamp et al. (1988) and Im et al. (2002). Cells were maintained in complete media, until reaching 90% confluence and then trypsinized, centrifuged, and resuspended in the same medium as described above. Primary keratinocytes from normal and DD subjects were grown in EpiLife medium supplemented with 0.06 mM Ca²⁺ and human keratinocyte growth supplement (Cascade Biologics, Portland, OR) as described previously (Foggia et al., 2006). HaCaT cells were infected using adenovirus encoding trpc1 gene or SERCA2-siRNA as described previously (Singh et al., 2001; Seth et al., 2004). Lanthanum chloride (LaCl₃) was obtained from Sigma-Aldrich (St. Louis, MO). Thapsigargin (Tg), 3,5-bistrifluoromethyl pyrazole (BTP2), and MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium] were obtained from Calbiochem (San Diego, CA), and isotretinoin (Accutane) was obtained from Hoffman-La Roche (Nutley, NJ).

mRNA Isolation, Synthesis of the First-Strand cDNA, and Reverse Transcription-Polymerase Chain Reaction (RT-PCR) Analysis
Total RNA was extracted from HaCaT cells by using TRIzol reagent (Invitrogen) and was treated with deoxyribonuclease I (1 U/µg RNA; Invitrogen) in a buffer containing 20 mM Tris-HCl, pH 8.4, 2 mM MgCl₂, and 50 mM KCl for 15 min at room temperature. The reaction was terminated by adding 2.5 mM EDTA and heating at 65°C for 10 min. mRNA was isolated using oligo(dT) column (QIAGEN, Valencia, CA). First-strand cDNA synthesis and RT-PCR were performed as described previously (Liu et al., 2000; Bollimuntha et al., 2005). After 30 cycles of amplifications, 10 µl of the RT-PCR product was analyzed on a 1% agarose gel, cloned into TA cloning vector (Invitrogen), and confirmed either by sequencing or restriction analysis.

Membrane Preparations and Western Blotting
HaCaT cells were harvested and lysed, and crude membranes were prepared from cell lysates (Singh et al., 2004). Skin samples (control and DD patients) were minced and treated with 10 µg/ml collagenase P (Hoffman-La Roche) for 15–30 min, followed by homogenization and membrane isolation as described in Liu et al. (2000) and Singh et al. (2004). Protein concentration was determined by using the Bradford method (Bio-Rad, Hercules, CA). Twenty-five micrograms of protein from crude membrane or whole cell lysates was resolved on 10% SDS-PAGE gels, transferred to polyvinylidene difluoride membranes, and probed with respective antibodies TRPC1 (Liu et al., 2000; Singh et al., 2001), SERCA2 (Calbiochem), TRPC3 (Singh et al., 2004), actin (Sigma-Aldrich), -tubulin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Santa Cruz Biotechnology, Santa Cruz, CA), and Apaf1, caspase-9, caspase-8, Bcl-xL, and NF-B-p65 subunit (Cell Signaling Technology, Beverly, MA) at (1:1000 dilution). Peroxidase-conjugated respective secondary antibodies were used to label the proteins. Proteins on the membrane were detected using West Pico-chemiluminescent substrate (Pierce Chemical, Rockford, IL) and analyzed either using Lumiimager (Hoffman-La Roche) or developing to x-ray films (Eastman Kodak, Rochester, NY).

Immunolocalization, Confocal Microscopy, and Hematoxylin and Eosin (H&E) Staining
Human and mouse skin samples were fixed using 5% paraformaldehyde, embedded in paraffin, and sectioned at 10-µm thickness. Individual sections were deparaffined in dimethylbenzene, rehydrated with a gradation of absolute alcohol through distilled water. Samples were blocked for 20 min with 5% donkey serum in phosphate-buffered saline (PBS) containing 0.5% bovine serum albumin (BSA) and incubated for 1 h with the desired primary antibodies (TRPC1, TRPC3; generously provided by Dr. Craig Montell, Department of Biological Chemistry, Johns Hopkins University, Baltimore, MD, and SERCA2 at 1:100 dilutions), washed three times, and incubated for 20 min with the desired fluorescein isothiocyanate (FITC) or rhodamine-conjugated secondary antibodies (1:100) as described in Liu et al. (2000). The secondary antibody was then removed by washing (3 times in PBS, 5 min each) and mounted on glass slides using Citifluor (Electron Microscopy Sciences, Hatfield, PA).

For immunofluorescence, HaCaT cells were grown overnight on coverslips, washed twice with PBS, and fixed for 30 min by using 3% paraformaldehyde. HaCaT cells used for NF-B-p65 nuclear translocation studies were grown on 35-mm glass-bottomed culture dishes (MatTek, Ashland, MA), stimulated with 1 µM thapsigargin for 30 min or 50 ng/ml tumor necrosis factor (TNF)- for 30 min before fixing, and permeabilized with 0.1% Triton X-100 in PBS. Cells were permeabilized using methanol for 5 min at –80°C and blocked as described above. For TRPC1, TRPC3, SERCA2, hemagglutinin (HA), and NF-B-p65 staining, cells were treated with the desired antibody at 1:100 dilutions for 1 h. Cells were washed (3 times with PBS 0.5% BSA) and probed with the required FITC- or rhodamine-conjugated secondary antibody (Jackson ImmunoResearch Laboratories, West Grove, PA) as described in Liu et al. (2000) and Singh et al. (2004). Fluorescence images were taken by using a confocal laser-scanning microscope (LSM 510 Meta; Carl Zeiss, Thornwood, NY). Fluorescence values for TRPC1, TRPC3, and SERCA2 staining were obtained using ImageJ software (National Institutes of Health, Bethesda, ND). For H&E staining, slides were washed, treated with hematoxylin for 5 min, and washed twice again with water. Slides were placed in 1% alcohol for 5–10 s, washed with water, and stained with eosin for 5 min. Slides were again washed twice dehydrated and mounted; images were taken using an LSM 510 Meta photomicroscope (Carl Zeiss).

Calcium Measurements
HaCaT cells were cultured on 35-mm glass-bottomed culture dishes (MatTek) for 24 h and incubated with 2 µM Fura-2 acetoxymethyl ester (AM) (Calbiochem) for 45 min at 37°C under an atmosphere of 5% CO₂, 95% air. The cells were washed three times with SES buffer with or without 1 mM Ca²⁺ (Liu et al., 2000). For fluorescence measurements, the fluorescence intensity of Fura-2 AM-loaded cells was monitored using a charge-coupled device camera-based imaging system (Compix, Cranberry, PA) mounted on an Olympus XL70 inverted microscope (Olympus America, Melville, NY) equipped with a Olympus 40x (1.3 numerical aperture) fluor objective. A monochrometer dual wavelength enabled alternative excitation at 340 and 380 nm, whereas the emission fluorescence was monitored at 510 nm with an Orka imaging camera (Hamamatsu, Bridgewater, NJ). The images of multiple cells collected at each excitation wavelength were processed using the Ca²⁺ imaging, PCI software (Compix) to provide ratios of Fura-2 fluorescence from excitation at 340 nm to that from excitation at 380 nm (F₃₄₀/F₃₈₀). Analog plots of the fluorescence ratio (340/380) in single cells are shown. Area under the curve for the total Ca²⁺ transients were calculated using SigmaPlot 8.0 (Systat Software, Point Richmond, CA).

Vybrant and Annexin-V Staining Assay
Vybrant apoptosis assay kit (Invitrogen) or Annexin-V-FLOUS staining kit (Hoffman-La Roche) were used to evaluate apoptosis as per manufacture's instruction. These kits distinguish apoptotic and necrotic cells by propidium iodide dye and YO-PRO-1 dye or FITC conjugated Annexin-V. The cells were visualized using a fluorescence microscope with 10x or 40x objectives, respectively. The dead and necrotic cells exhibit red fluorescence of the nucleus along with the green fluorescence, whereas apoptotic cells have a characteristic green fluorescence. The numbers of green cells against total number of cells (differential interference contrast; DIC) were counted for each experiment, and the values are represented in terms of number of apoptotic cells/100 cells counted. The values shown are mean of five individual experiments performed in duplicate.

Cell Viability (MTT) Assay
HaCaT cells were seeded on 96-well plates at a density of 5 x 10⁴ cells/well. Cell viability was determined by MTT assay, performed in triplicates, as described previously (Bollimuntha et al., 2005). After incubation with the desired drug, 100 µl of MTT reagent (0.5 mg/ml MTT in SES buffer containing 10 mM HEPES, 145 mM NaCl, 5 mM KCl, 1 mM MgCl₂, 1 mM CaCl₂, and 10 mM glucose, pH 7.4) supplemented with 10% FBS was added to each well and incubated in a CO₂ incubator for 2–4 h. The medium was aspirated from each well, and the culture plate was dried at 37°C for 1 h. The resulting formazan dye was extracted with 100 µl of 0.04 N HCl in isopropanol, and the absorbance was measured in a microplate reader (Molecular Devices, Sunnyvale, CA) at 570 nm with a background correction at 650 nm. To determine the proliferation of primary keratinocytes, cells from normal (passages 4 and 5) and DD subjects (passages 4, 5, and 7) were seeded at a density of 2 x 10⁴ cell/well in 96-well plates. Forty-eight hours postseeding MTT assay was performed as mentioned above, except 0.5 mg/ml MTT reagent was made in the EpiLife medium in which the cells were grown.

Dual-Luciferase Reporter Assay
HaCaT cells were grown to 60% confluence in 12-well plates. Transfection of reporter constructs was done using Lipofectamine 2000 (Invitrogen), as per manufacturer's instructions. Reporter vector mixture containing 1.5 µg of pNFkB-Luc (containing firefly luciferase gene from Photinus Pyralis; Clontech, Mountain View, CA) and 0.06 µg of pRL-TK (containing Renilla luciferase gene from Renilla reniformis, used as internal control; Promega, Madison, WI) incubated with required amount Lipofectamine 2000 reagent in serum-free DMEM was used per well for transfection. Complete DMEM with or without 10 multiplicity of infection (MOI) of adenovirus expressing SERCA-siRNA was supplemented at 4 h after transfection. After 36–40 h of transfection, HaCaT cells were stimulated with 1 µM Tg or 50 ng/ml TNF- for another 4 h. HaCaT cells were treated for 30 min at 37°C with 100 nM of an NF-B activation inhibitor (catalog no. 481407; Calbiochem), before stimulation. After stimulation, cells were washed twice with ice-cold PBS and lysed with 200 µl of the supplied lysis buffer. Dual luciferase assay was performed following manufacturer's instructions (Promega). A 20-µl aliquot was taken for measuring the respective luciferase activities using a Zylux Femtomaster FB12 luminometer. Protein concentrations were determined using Bio-Rad reagents. The normalized reporter activities, indicating relative luciferase units per microgram of protein, were expressed as -fold induction in luciferase activity.

Statistics
Data analysis was performed using Origin 7.0 (OriginLab, Northampton, MA). Statistical comparisons were made using the two-tailed Student's t test. Experimental values are expressed as mean ± SEM. Differences in the mean values were considered to be significant at p < 0.05.

	RESULTS

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TRPC1 Is Up-Regulated in DD Patients and SERCA2^+/– Mice
The expression of TRPC channels in the epidermis of control and DD patients was investigated. Immunostaining of control human skin samples using TRPC1 or SERCA2 antibody resulted in immunoreactivity in the epidermal layers (Figure 1A). Both TRPC1 and SERCA2 were expressed in the proliferating layers of the skin and showed colocalization (Figure 1, A and B). However, TRPC1 immunoreactivity in DD patients was significantly increased (1.5-fold; fluorescence values were 964 ± 81 and 609 ± 65, respectively, as obtained using an ImageJ software, in DD and control tissues; these values are averages of 3 independent experiments) (Figure 1E). In contrast, staining with SERCA2 antibody showed a decrease in its immunoreactivity in DD patients (870 ± 92 compared with control 939 ± 80; these values were obtained using ImageJ software) (Figure 1E). Overlay of both TRPC1 and SERCA2 showed colocalization in the skin sections of DD patients (Figure 1B). Interestingly, significant TRPC1 staining was observed in the generative layer of epidermis, important for cell proliferation. It is important to note that down-regulation of SERCA2 in cardiac myocytes was shown to increase TRPC4, and TRPC5 expression; however, expression of TRPC1 was not measured in that study (Seth et al., 2004). We also investigated whether TRPC3 level was changed in DD epidermal tissue. As shown in Figure 1, C and D, no significant increase in TRPC3 immunoreactivity was observed (792 ± 62 compared with control 813 ± 51) or in SERCA2 (as seen in Figure 1, A and B). Although other TRPC expression has not been assessed as yet in DD tissue, these data suggest that DD might induce specific effects on different TRPC channels.

View larger version (86K): [in this window] [in a new window]   Figure 1. TRPC1 and TRPC3 expression in control and DD patients. Immunofluorescence in skin sections (10 µm) obtained from control (A and C) or DD patient (B and D), using anti-TRPC1 (A and B), anti-SERCA2, and anti TRPC3 (C and D) at 1:100 dilutions. Fluorescently labeled secondary antibodies were used to label TRPC1, TRPC3, and SERCA2 proteins. Images were taken using 63x objective. (E) Bar graph indicating immunofluorescence values of SERCA2, TRPC1, and TRPC3 in control and DD patients. (F) Western blot using crude membranes prepared from control and DD patients and probed using TRPC1, SERCA2, and actin antibodies. (G) Images (H&E staining and DIC) from control and DD patients.

To confirm the results obtained with DD patients who have only one normal copy of the gene encoding SERCA2, we used epidermal tissue from SERCA2^+/– mice, which like DD patients have one normal copy of SERCA2 gene. The SERCA2 gene was targeted previously by removing the promoter and first two exons, which eliminated expression of the mutant gene (Periasamy et al., 1999). Immunolocalization was performed on skin samples from SERCA2^+/+ and SERCA2^+/– mice. Similar to the results with DD tissue, TRPC1 protein was up-regulated in the skin sections of SERCA2^+/– mice (Figure 2A, red fluorescence), whereas there was no increase in TRPC3 levels (our unpublished data). Importantly, increased TRPC1 expression was observed in mouse epidermal layers, which is consistent with the results obtained from DD patients. Expression of SERCA2 in the skin cells of SERCA2^+/– mice was also reduced compared with control (Figure 2A, green fluorescence). Moreover, Western blots performed on skin samples obtained from SERCA2^+/+ and SERCA2^+/– mice showed a decreased expression of SERCA2 in SERCA2^+/– mice, whereas no change in actin levels was observed (Figure 2B). Overall, these results suggest that a loss of function and/or reduced expression of SERCA2 increase TRPC1 expression in epidermal keratinocytes.

View larger version (57K): [in this window] [in a new window]   Figure 2. SERCA2+/– mice exhibit increased TRPC1 expression. (A) Immunofluorescence in skin sections (10 µm) obtained from SERCA2+/+ or SERCA2+/– mice using anti-TRPC1 and anti-SERCA2 antibodies. Fluorescently labeled secondary antibodies were used to label TRPC1 and SERCA2 proteins. Images were taken using 63x objective. (B) Western blots performed on samples obtained from SERCA2+/+ and SERCA2+/– mice and probed using SERCA2 and -actin antibodies.

View larger version (77K): [in this window] [in a new window]   Figure 3. Expression of TRPC1 and TRPC3 in HaCaT cells. (A) RT-PCR on HaCaT cells using trp1- and trp3-specific primers (top) or GAPDH primers (middle) as described in Bollimuntha et al. (2005) and Kobayashi et al. (2003), bottom, is control PCR using plasmid DNA. (B) Western blot on crude membranes prepared from HaCaT cells and probed using TRPC1 or TRPC3 antibodies. Control samples were obtained from HSG cells expressing endogenous TRPC1 and 293 cells expressing endogenous TRPC3. (C) Localization of endogenous TRPC1 and TRPC3 in HaCaT and SERCA2-siRNA–expressing cells using TRPC1 and TRPC3 antibodies followed by rhodamine-conjugated secondary antibodies. Control indicates staining without TRPC1 or TRPC3 antibodies but treated with rhodamine-labeled secondary antibody.

Localization of endogenous TRPC1 and TRPC3 proteins in HaCaT cells was determined by immunofluorescence. Control HaCaT cells were cultured on glass coverslips for 24 h, and TRPC1 and TRPC3 proteins were detected using polyclonal anti-TRPC1 and -TRPC3 antibodies. Punctate staining of TRPC1 protein was observed along the plasma membrane and in the subplasma membrane region, further confirming the expression of TRPC1 in these cells (Figure 3C). No immunoreactivity was observed in the absence of the primary antibody (Figure 3C). TRPC3 was also detected in the plasma membrane region, along with some subplasma membrane staining (Figure 3C). These results are consistent with our previous results, demonstrating localization of TRPC1 and TRPC3 in the plasma membrane (Liu et al., 2000; Singh et al., 2004) and with another report, where TRP proteins were shown to be expressed in the plasma membrane of normal human keratinocytes (Tu et al., 2005). Moreover, localization of TRPC1 and TRPC3 was not altered in HaCaT cells overexpressing SERCA2-siRNA (Figure 3C). Together, the data presented above suggest that TRPC1 and TRPC3 are expressed in human keratinocytes and can potentially contribute toward regulation of Ca²⁺ entry and cytosolic [Ca²⁺]. However, because only TRPC1 was up-regulated in DD, the role of this channel in keratinocyte growth was further examined.

SERCA2 Gene Silencing Increases Thapsigargin-stimulated Ca²⁺ Influx and TRPC1 Expression Levels
Keratinocytes from DD patients have only one normal copy of SERCA2. It has been hypothesized that a single copy of the gene is insufficient to encode adequate amount of SERCA protein, and significant changes in cellular Ca²⁺ regulation and cell growth are seen in these cells (Sakuntabhai et al., 1999; Ahn et al., 2003; Foggia et al., 2006). To examine the possible consequences of SERCA2 hypofunction, we knocked down SERCA2 in HaCaT cells using adenovirus encoding either SERCA2-siRNA or control-siRNA. HaCaT cells were transiently infected with the required adenovirus and Ca²⁺ imaging was performed on cells stimulated with Tg. As shown in Figure 4A, addition of Tg in a Ca²⁺-containing media, induced an increase in cytosolic Ca²⁺ ([Ca²⁺]_i), which was significantly higher in HaCaT cells expressing SERCA2-siRNA, whereas no increase in control-siRNA was observed (Figure 4A). Mean Ca²⁺ influxes (area under the curve) are shown in Figure 4D. Importantly, release of Ca²⁺ from the internal stores (measured upon Tg addition in the absence of external Ca²⁺) was significantly lower in cells overexpressing SERCA2-siRNA, whereas no change was observed in TRPC1-overexpressing cells (Figure 4B). TRPC1 or SERCA2-siRNA expressing cells were also stimulated with a muscarinic agonist carbachol, which showed a significant increase in Ca²⁺ influx (Figure 4C). Overexpression of TRPC1 increased Tg-stimulated [Ca²⁺]_i influx was also significantly decreased upon isotretinoin treatment (a medication that improves DD symptoms; Figure 4D).

View larger version (39K): [in this window] [in a new window]   Figure 4. Ca2+ influx in HaCaT cells and DD keratinocytes. (A) Tg-stimulated fluorescence trace in HaCaT cells transiently expressing adenovirus encoding control or SERCA2-siRNA (MOI of 10 plaque-forming units/cell) in a Ca2+ containing SES media. (B) Traces of intracellular release (mean traces from >50 cells each). (C) Fluorescence traces of control or cells expressing SERCA2-siRNA or TRPC1 upon carbachol stimulation. (D) Ca2+ influx values measured as area under the curve; number of cells imaged also is labeled. (E) Traces (mean value of 50–60 cells) in cells pretreated with 1 mM lanthanum for 15 min or 1 µM SOCE blocker BTP2 for 60 min. (F) Tg-stimulated fluorescence trace in keratinocytes isolated from normal and DD patients. (G) Western blots using SERCA2, TRPC1, and actin antibodies and representative of three to five individual experiments (quantification added to the blot). Asterisk (*) indicates mean values that are significantly different (p < 0.05).

Western blot analysis performed on HaCaT cells transiently overexpressing SERCA2-siRNA confirmed a decrease in SERCA2 expression (quantification of bands indicate 65% decrease in SERCA2 protein levels; Figure 4G). Furthermore, consistent with the data shown in Figure 1 expression of SERCA2-siRNA–treated cells displayed increase in TRPC1 expression (Figure 4G, quantification of bands indicate 40% increase in TRPC1 protein levels, no significant change in actin protein was observed). Moreover, TRPC1 protein levels were unaffected upon isotretinoin treatment (100 µM for 12 h) of control or SERCA2-siRNA expressing HaCaT cells (Figure 4G, right). These results suggest that down-regulation of SERCA2 in keratinocytes alters cellular Ca²⁺ homeostasis, resulting in increased Ca²⁺ entry. We suggest that Ca²⁺ entry can be attributed to the increase in TRPC1 expression. However, we presently do not understand how isotretinoin modulates Ca²⁺ entry in Tg-treated HaCaT cells.

TRPC1 Overexpression Promotes Cell Survival by Inhibiting Apoptosis
To investigate the effect of altering SERCA2 on cell viability, we examined Tg-induced apoptosis in HaCaT cells. As shown in Figure 5A, control HaCaT cells showed little cell death (YO-PRO-1 staining, 2 cells stained/100 cells, average data are shown in B). In contrast, cells treated with Tg for 30 min showed a significant increase in cell death (Figure 5, A and B; Bollimuntha et al., 2005, 30 cells/100 cells, respectively), which did not seem to be dependent on external Ca²⁺. Strikingly, TRPC1 overexpression significantly decreased Tg-induced cell death (80% reduction, 5 cells/100 cells) in normal Ca²⁺-containing medium, more dye uptake was seen in Tg-treated cells in the absence of external Ca²⁺ (Figure 5, A and B). Addition of necrotic dye propidium iodide showed no staining under these conditions (our unpublished data); suggesting that Tg induces apoptosis mediated cell death.

View larger version (67K): [in this window] [in a new window]   Figure 5. Overexpression of TRPC1 restricts Tg-mediated apoptosis. Marker for apoptosis (YO-PRO-1) was used to stain control and TRPC1-overexpressing cells as described in Bollimuntha et al. (2005). Tg (1 µM) was treated for 0 or 30 min (A). Fluorescence images of cells stained with YO-PRO-1 were taken using 10x or 40x objective, and green cells were counted along with total number of cells. (B) Fluorescence images of Annexin-V–positive cells taken immediately using 10x or 40x objectives, and green cells were counted along with total number of cells. (C) Represents mean bar graph from >1000 cells in each group. (D) Represents mean bar graph from >800 cells in each group. (E) Western blot on crude membranes and whole extracts prepared from HaCaT cells or TRPC1-overexpressing cells and probed using TRPC1, Bcl-xL, caspase-8, caspase-9, Apaf-1, and GAPDH antibodies. Asterisk (*) indicates values significantly different from its counterpart (p < 0.05).

To confirm these results, surviving cells were determined by MTT assay. There was a dose-dependent decrease in cell survival upon Tg treatment (30 min) (Figure 6A). Consistent with the protection against apoptosis, SERCA2-siRNA treatment increased cell survival after Tg treatment (Figure 6A). Keratinocytes overexpressing TRPC1, but not TRPC3, showed similar increases in cell survival. However, treatment with isotretinoin further decreased cell survival after treatment with 1 µM Tg, which was again increased upon expression of either TRPC1 or SERCA2-siRNA but not TRPC3 (Figure 6B). To assess the transfection efficiencies, confocal studies were performed on cells overexpressing HA-TRPC1 or SERCA2-siRNA. As indicated in Figure 6C, staining of HaCaT cells using HA antibody demonstrated a significant expression (90% cells were positive with HA antibody). Transfection with SERCA2-siRNA showed 90% transfection, and consistent with this, there was a significant reduction in the SERCA2 protein level compared with the level of SERCA2 in control cells (Figure 6C). Similar SERCA2-siRNA treatment has been used to effectively decrease SERCA2 protein in rat and chicken cardiac myocytes (Seth et al., 2004). Interestingly, overexpression of TRPC1 or SERCA2-siRNA expression increased the number of MTT-positive cells even in cells that were not treated with Tg (Figure 6B). In contrast, cells treated with isotretinoin showed a decreased survival (Figure 6B). Addition of BTP2 or La³⁺ (TRPC1 inhibitors) significantly decreased TRPC1-mediated cell proliferation in HaCaT cells (Figure 6D). Stimulation of cells with muscarinic agonist carbachol also increased cell survival, which was significantly higher in cells overexpressing TRPC1 or SERCA2-siRNA (Figure 6E). Moreover, DD keratinocytes also showed significant increase in cell survival both in the presence and absence of Tg (Figure 6F). Proliferation of normal and DD keratinocytes was sensitive to isotretinoin treatment (Figure 6F). These results are consistent with previous studies where TRPC1 inhibited translocation of proapoptotic proteins from the ER to mitochondria in neuronal cells (Bollimuntha et al., 2005), and Ca²⁺ influx inhibitors decreased cell proliferation (Benzaquen et al., 1995) and enhanced apoptosis (Gill et al., 1996; Enfissi et al., 2004).

View larger version (49K): [in this window] [in a new window]   Figure 6. Overexpression of SERCA2-siRNA or TRPC1 increases cell proliferation. (A) MTT assays performed on control, TRPC1-, TRPC3-, and SERCA2-siRNA–overexpressing cells treated with 1 µM Tg for 30 min in the absence (left) or presence of 100 µM isotretinoin, 12 h (right) with or without Ca2+. (B) Cell survival (MTT assay) on control, TRPC1-, and SERCA2-siRNA–overexpressing cells in the presence of isotretinoin. (C) Fluorescence images on HaCaT cells overexpressing HA-TRPC1 or endogenous SERCA2, or cells expressing SERC2 siRNA. TRPC1 was stained using HA antibody (1:500), whereas SERCA2 was stained using SERCA2 antibody (1:1000). Proteins were detected using FITC-labeled specific secondary antibodies. (D) Cell survival in control and TRPC1-overexpressing HaCaT cell treated with SOCE inhibitor BTP2 (1 µM) and 1 mM La3+ for 12 h. (E) Cell proliferation upon 100 µM carbachol stimulation for 12 h. (F) Cell survival of keratinocytes isolated from normal and DD patients, respectively, in presence of 1 µM Tg or 100 µM isotretinoin for 12 h. Asterisk (*) indicates values significantly different from its counterpart (p < 0.05).

TRPC1-mediated Cell Survival Is Dependent on NF-B Activation

View larger version (40K): [in this window] [in a new window]   Figure 7. SERCA2-siRNA or TRPC1-mediated protection depends on NF-B stimulation. (A) Fluorescence images on control and TRPC1-expressing cells treated with 1 µM Tg for 30 min and stained using p65 antibody and 4,6-diamidino-2-phenylindole (DAPI) (to counterstain nucleus). Average data are shown as bar graph. Asterisk (*) indicates values significantly different from its counterpart (p < 0.05). (B) TNF- (50 ng/ml; 4 h) or Tg (1 µM; 4 h) stimulated activation of NF-B luciferase reporter construct in control and SERC2-siRNA–expressing HaCaT cells, in the absence or presence of 100 nM NF-B inhibitor. (C) Cell survival (MTT assay) on control, TRPC1, and SERCA2-siRNA–overexpressing cells in the absence or presence of 100 nM NF-B inhibitor for 12 h and extracellular Ca2+, respectively. (D) Western blots on control and NF-B inhibitor (100 nM; 12 h)-treated HaCaT cells using TRPC1, SERCA2, and -tubulin antibodies.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Here, we report for the first time that TRPC1 protein is up-regulated, in keratinocytes from DD patients. TRPC1 is also up-regulated in epidermal layers obtained from SERCA^+/– mice, an animal model for DD. Moreover TRPC1 expression in squamous layers could be critical for growth and proliferation of keratinocytes. Similar up-regulation of TRPC1 is seen when SERCA2 expression is decreased by expression of SERCA2-siRNA in a human keratinocyte cell line (HaCaT). The effect is relatively specific, because expression of TRPC3 is not altered. Down-regulation of SERCA2 in HaCaT cells or keratinocytes obtained from DD patients significantly increased Ca²⁺ influx upon stimulation with Tg without any change in basal Ca²⁺ influx. Thapsigargin-mediated Ca²⁺ release from the internal stores was also decreased in SERCA2-siRNA–expressing cells or in DD keratinocytes, suggesting a decreased Ca²⁺ content in the ER store, as a result of compromised SERCA2 activity. Addition of isotretinoin, a drug used for temporary symptomatic relief of DD, induces a significant decrease in Ca²⁺ influx in SERCA2-siRNA–expressing cells without affecting internal Ca²⁺ release or TRPC1 protein level. Isotretinoin also decreased Tg-stimulated Ca²⁺ entry in cells overexpressing TRPC1. Taken together, these results demonstrate that down-regulation of SERCA2 significantly affects plasma membrane Ca²⁺ regulation. Notably, a major consequence of these disruptions in ER Ca²⁺ signaling is increased Ca²⁺ entry due to an increase in TRPC1 expression. This leads to higher intracellular [Ca²⁺] after treatment with Tg.

A key finding of this study is that overexpression of TRPC1, either directly or via SERCA2-siRNA treatment, in HaCaT cells confers resistance to Tg-mediated apoptosis and increases cell survival (Figure 8). Whereas, overexpression of TRPC3 did not show any increase in cell survival. Furthermore, the protective effect of TRPC1 was associated with the presence of external Ca²⁺, suggesting a role of Ca²⁺ entry via TRPC1 channel in cell survival. Our study also indicates that elevation of cytosolic Ca²⁺ as a result of increased SOCE is not associated with apoptosis; instead, it promotes activation of NF-B pathway leading to an antiapoptotic effect. Addition of NF-B inhibitors significantly decreased TRPC1 levels and also inhibited TRPC1-mediated cell proliferation, which was dependent on extracellular Ca²⁺. This indicates that NF-B–induced TRPC1 expression is critical for cell proliferation/survival. TRPC1 and other members of its family, involved in SOCE, have been associated with cell proliferation and the level of expression of different TRP proteins has been linked with the proliferative capacity of different cell lineages (Pla et al., 2005). SOCE is implicated in activating signaling cascades, influencing cell division and promoting cell proliferation (Berridge et al., 2000; Golovina et al., 2001). It has been also reported that Ca²⁺ is important for activating the transcription factor NF-B (Miyamoto et al., 1998; Tabary et al., 2006) and that activation of NF-B followed by the nuclear translocation of its transactivating p65 subunit is critical for regulation of apoptosis and cell proliferation (Lilienbaum and Israel, 2003). Increase in Bcl-xL expression in TRPC1 up-regulated cells further substantiates its transcriptional regulation by NF-B (Glasgow et al., 2001; Marinari et al., 2004). Increased expression of Bcl-xL would shift the ratio of pro- and antiapoptotic machinery in favor of cell survival; additionally, it would also work at the level of ER and mitochondria in regulating Ca²⁺ handling and restoring membrane potential upon exposure to apoptotic stimuli. Interestingly, histology of DD skin biopsies also showed hyperproliferative clusters of cells within the epidermis (Lucker et al., 1996). Moreover, it has been shown in vitro that Ca²⁺ is important for keratinocyte differentiation and development of intercellular junctions (Hennings et al., 1980; Elias et al., 2002). Based on our observation that TRPC1 is overexpressed in the proliferative layer of the epidermis, we suggest that TRPC1-induced resistance to apoptosis might play a role in the abnormal keratosis associated with this disease. Interestingly, Foggia et al. (2006) also showed that capacitative Ca²⁺ influx was increased in DD patients; however, cytosolic Ca²⁺ was decreased as a result of overexpressed ATP2C1 (a Golgi Ca²⁺ pump). These results complement our data and indicate perhaps ATP2C1 and TRPC1 could work together to maintain Ca²⁺ homeostasis in long-term DD condition; however, it remains to be investigated whether TRPC1 regulate or interact with ATP2C1 protein. Together with previous studies showing up-regulation of TRPC4 and TRPC5 after SERCA2 knockdown in myocytes (Seth et al., 2004), the present data reveal an interesting communication between the ER and plasma membrane. One possible role for increased TRPC1 expression after loss of SERCA2 would be to increase Ca²⁺ entry in order to maintain [Ca²⁺]_i. In addition, the novel data reported here demonstrate that TRPC1 up-regulation exerts a prosurvival effect on keratinocytes. Thus, remodeling of Ca²⁺ signaling pathways can compensate for the aberrant SERCA2 function and the resulting deleterious consequences on cell growth and survival via upregulation of "prosurvival" proteins. Although, there are likely to be multiple genes that are altered under these conditions, our data provide a novel "cross-talk" between SERCA2 and TRPC1 and that the antiapoptotic message generated from this communication upon cell stress is relayed to the nucleus, at least in part, via the NF-B mechanism (Figure 8). SERCA2 down-regulation has been shown to induce Ca²⁺-dependent transcriptional and nuclear factors activity (Seth et al., 2004). Further studies in this regard will be required to determine the exact mechanisms by which SERCA2 down-regulation leads to the up-regulation of TRPC1 proteins. For example it would be important to determine whether SERCA2-associated transcription factors are also involved in TRPC1 gene expression. Another important question that remains to be elaborated is how exactly up-regulation of TRPC1 would lead to an increase in cell survival and resistance to apoptosis. Such information will be useful in understanding the mechanisms involved in hypertrophy and hyperplasia and defining the role of TRPC channels in these processes.

View larger version (34K): [in this window] [in a new window]   Figure 8. Proposed model for the role of TRPC1 in Darier's disease: We propose that in Darier's disease keratinocytes, the nonfunctional SERCA fails to maintain the adequate ER calcium pool (, compromised function). This decreased SERCA activity in turn up-regulates the store-operated plasma membrane calcium channel TRPC1, in an undefined mechanism dependent on NF-B. Overexpressed TRPC1 channels augment store-operated calcium influx. The incoming calcium is pumped out via the plasma membrane calcium ATPase (PMCA) and also used for the cells physiological need. An optimal level of this calcium brings about proper growth and proliferation (, positive effect) by activating NF-B pathway. This confers resistance to apoptosis by regulating the expression of proproliferative/antiapoptotic genes. However, if there is an overload this calcium might be detrimental, leading to stress-induced apoptosis or uncontrolled proliferation accounting for cancerous state (?). It is also proposed that isotretinoin, by yet another undefined mechanism (?) regulates the calcium influx via TRPC1 and blocks the calcium overload, thereby inhibiting hyperproliferation and probably cancer.

	ACKNOWLEDGMENTS

 
We thank Drs. Giuseppe Inesi, Craig Montell, Greg Barritt, Jose Enrique Mejia, Min Wu, Shmuel Muallem, Gary Stull, and Gene Homandberg for reagents, discussion, and support. We especially thank Ashley Bansal, Sunitha Bollimuntha, and Tami Casavan for technical assistance with imaging facility and cell culture. This work was supported by National Science Foundation Grant 0548733 and National Institutes of Health Grants DE 017102 and 5P20RR017699 (to B.B.S.).

	Footnotes

 
This was published online ahead of print in MBC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E06-03-0251) on July 26, 2006.

Address correspondence to: Brij B. Singh (bsingh{at}medicine.nodak.edu)

Abbreviations used: DD, Darier's disease; SERCA, sarco(endo)plasmic reticulum Ca²⁺ ATPase; SOCE, store operated calcium entry; Tg, thapsigargin; TRPC1, transient receptor potential canonical 1.

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