Epithelial TRPV1 Signaling Accelerates Gingival Epithelial Cell Proliferation

Abstract

Transient receptor potential cation channel subfamily V member 1 (TRPV1), a member of the calcium-permeable thermosensitive transient receptor potential superfamily, is a sensor of thermal and chemical stimuli. TRPV1 is activated by noxious heat (> 43°C), acidic conditions (pH < 6.6), capsaicin, and endovanilloids. This pain receptor was discovered on nociceptive fibers in the peripheral nervous system. TRPV1 was recently found to be expressed by non-neuronal cells, such as epithelial cells. The oral gingival epithelium is exposed to multiple noxious stimuli, including heat and acids derived from endogenous and exogenous substances; however, whether gingival epithelial cells (GECs) express TRPV1 is unknown. We show that both TRPV1 mRNA and protein are expressed by GECs. Capsaicin, a TRPV1 agonist, elevated intracellular Ca²⁺ levels in the gingival epithelial cell line, epi 4. Moreover, TRPV1 activation in epi 4 cells accelerated proliferation. These responses to capsaicin were inhibited by a specific TRPV1 antagonist, SB-366791. We also observed GEC proliferation in capsaicin-treated mice in vivo. No effects were observed on GEC apoptosis by epithelial TRPV1 signaling. To examine the molecular mechanisms underlying this proliferative effect, we performed complementary (c)DNA microarray analysis of capsaicin-stimulated epi 4 cells. Compared with control conditions, 227 genes were up-regulated and 232 genes were down-regulated following capsaicin stimulation. Several proliferation-related genes were validated by independent experiments. Among them, fibroblast growth factor-17 and neuregulin 2 were significantly up-regulated in capsaicin-treated epi 4 cells. Our results suggest that functional TRPV1 is expressed by GECs and contributes to the regulation of cell proliferation.

Introduction

Transient receptor potential cation channel subfamily V member 1 (TRPV1), also known as the capsaicin receptor or vanilloid receptor 1, is a non-selective ligand-gated cation channel activated by a range of exogenous and endogenous physical and chemical stimuli (Caterina et al., 1997). Noxious heat (> 43°C), acidic conditions (pH < 6.6), and capsaicin, the pungent compound present in red chilies, are prototypical activators of TRPV1 (Dhaka et al., 2006). TRPV1 was originally identified on peripheral nociceptive fibers, where its activation evokes pain or discomfort and initiates reflexes to protect the host (Lin et al., 2007). Recently, TRPV1 channels were also found to play a sensory role in non-neuronal cells, such as keratinocytes (Peier et al., 2002), gastric epithelial cells (Lee et al., 2007), and urinary bladder epithelial cells (Birder, 2005). Other studies revealed that the activation of epithelial TRPV1 affects numerous biological processes, including proliferation, differentiation, and apoptosis (Ip et al., 2012; Liu et al., 2012).

Gingival epithelial cells (GECs) contribute to homeostasis in periodontal tissues by forming a physical barrier protecting against exogenous noxious agents. In addition, GECs sense and respond to bacterial stimuli by activating pathogen recognition receptors, including Toll-like and nucleotide-binding oligomerization domain (NOD)–like receptors (Ji et al., 2009). GECs are also exposed to physical and chemical stimuli, including high temperatures, mechanical pressure, and acids derived from foods, microbes, and inhaled antigens. However, the expression of TRPV1 as an environmental sensor and its physiological functions in GECs are poorly understood.

In this study, we hypothesized that TRPV1 is expressed by GECs and is involved in cellular functions in the gingival epithelium. We examined the expression of TRPV1 mRNA and protein in both human and mouse GECs. We investigated the cellular functions mediated by TRPV1 in GECs in vitro and in vivo. Finally, we explored the downstream effectors of TRPV1 signaling by cDNA microarray analysis.

Materials & Methods

Reagents and Antibodies

Capsaicin (≥ 95% purity, from Capsicum sp.) was purchased from Sigma-Aldrich Corporation (St. Louis, MO, USA). SB-366791 was purchased from Focus Biomolecules (Plymouth Meeting, PA, USA). Anti-TRPV1 antibody for immunostaining was obtained from Alomone Labs (Jerusalem, Israel). Rabbit anti-mouse glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) and peroxidase-labeled anti-rabbit antibody (GE Healthcare, Little Chalfont, Buckinghamshire, UK) were used for Western blotting experiments.

Cell Preparation and Culture

Prior to inclusion in this study, all human participants provided written informed consent according to a protocol that was reviewed and approved by the Institutional Review Board of the Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan. Human GECs were prepared from clinically normal gingival tissue obtained following the extraction of an uninfected third molar, as previously described (Takahashi et al., 2010). The simian virus 40 (SV40)-immortalized human gingival epithelial cell line, epi 4, was established and maintained as previously described (Murakami et al., 2002; Takahashi et al., 2011). Human embryonic kidney (HEK)-293 cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 µg/mL streptomycin.

The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and Wound-healing Assays in vitro

The MTT assay was performed according to the manufacturer’s (Sigma-Aldrich Corporation) instructions for analyzing proliferation. For the wound-healing assay, epi 4 cells were grown in 12-well plates, and a small linear scratch was created in the confluent monolayer by gentle scraping with sterile pipette tips. Photographs of wound closure were taken at the end of the experiment and analyzed with ImageJ software (National Institutes of Health, Bethesda, MD, USA), and the relative gap closure was measured.

Mice

All experiments were performed in accordance with the Regulations and Guidelines on Scientific and Ethical Care and Use of Laboratory Animals of the Science Council of Japan and were approved by the Institutional Animal Care and Use Committee at Niigata University, Niigata, Japan. Six- to eight-week-old male C57BL/6 mice were obtained from Japan SLC, Inc. (Shizuoka, Japan), maintained under specific pathogen-free conditions, and fed regular chow and sterile water.

Immunohistochemistry

Fixed mandibles were dissected, decalcified, embedded, and sectioned as previously described (Sulniute et al., 2011), with minor modifications. Paraffin sections were incubated with anti-TRPV1 antibody overnight. Immunoreactivity was detected with biotinylated chicken anti-rabbit immunoglobulin (IgG) (Abcam, Cambridge, UK) in an avidin-biotin-immunoperoxidase system (Vector Laboratories, Inc., Burlingame, CA, USA). Counterstaining was performed with hematoxylin (Polysciences, Inc., Warrington, PA, USA). For the immunohistochemistry of cultured cells, epi 4 cells were seeded in a Lab-Tek™ Chamber Slide (Nunc, Rochester, NY, USA) at a density of 5 × 10⁴ cells/well. The attached cells were partially fixed with chloroform/acetone, washed in phosphate-buffered saline (PBS), and stained with anti-TRPV1 antibody. The sections were then imaged by microscopy (Biozero BZ-8000; Keyence Corporation, Osaka, Japan).

Transfection of a TRPV1 Overexpression Vector

A TRPV1 overexpression plasmid was kindly provided by Dr. Ardem Patapoutian (Scripps Research Institute, La Jolla, CA, USA). The HEK-293 cells were transfected with Lipofectamine^® 2000 (Life Technologies, Carlsbad, CA, USA), according to the manufacturer’s instructions. TRPV1 expression was confirmed by conventional PCR and Western blotting at 24 hr post-transfection.

Polymerase Chain-reaction (PCR) and Gel Electrophoresis

Total RNA was isolated from gingival tissues and cells with TRI Reagent^® (Molecular Research Center, Inc., Cincinnati, OH, USA). cDNA was synthesized with Transcriptor Universal cDNA Master (Roche Molecular Systems, Inc., Branchburg, NJ, USA). Conventional PCR was performed with a GeneAmp^® PCR System 7700 (Applied Biosystems, Carlsbad, CA, USA), and PCR products were run on 1.5% agarose gels and visualized with SYBR^® Safe DNA (Invitrogen Corporation, Carlsbad, CA, USA). Quantitative PCR was performed on a LightCycler® 480 (Roche Molecular Systems) with EagleTaq Master Mix (Roche Molecular Systems). The relative expression level of each mRNA was normalized to that of GAPDH mRNA by the delta delta Ct method (Livak and Schmittgen, 2001). The custom-designed oligonucleotide sequences (Invitrogen Corporation) used for both conventional and quantitative PCR are summarized in the Appendix Table.

Western Blotting

Total protein was extracted with M-PER Mammalian Protein Extraction Reagent (Thermo Fisher Scientific, Inc., Rockford, IL, USA). Protein concentration was determined with a Pierce Bicinchoninic Acid Protein Assay Kit (Pierce Biotechnology, Inc., Rockford, IL, USA). Each sample was solubilized in sodium dodecyl sulfate (SDS) sample buffer, separated by SDS-polyacrylamide gel electrophoresis, and transferred to polyvinylidene fluoride membranes (EMD Millipore Corporation, Billerica, MA, USA). After incubation with each antibody, target proteins were detected with ECL Plus Western blotting detection reagents (GE Healthcare) and a LumiVision PRO 400EX system (Aisin Seiki Co., Ltd., Aichi, Japan).

Ca²⁺ Influx Assay

Intracellular Ca²⁺ changes were examined with the Calcium Kit II-Fluo 4 (Dojindo Laboratories, Tokyo, Japan), according to the manufacturer’s instructions. In brief, cells seeded in 96-well plates were treated with loading buffer containing Fluo4-AM at 37°C for 1 hr. Following replacement of the buffer with the recording medium, fluorescent intensity was measured, 1 min after stimulation, with a microplate fluorometer (TriStar LB 941; Berthold Technologies GmbH & Co. KG, Bad Wildbad, Germany).

Bromodeoxyuridine (BrdU) Labeling in vivo

The BrdU labeling was used to examine GEC proliferation in vivo. Mice were injected intraperitoneally with 100 mg/kg of BrdU (BD Pharmingen, San Jose, CA, USA) in PBS. Maxillary jawbones from euthanized mice were dissected 72 hr after BrdU injection, and fixed, decalcified, then embedded in paraffin. BrdU staining was performed with the BrdU in situ detection kit (BD Pharmingen), according to the supplier’s recommendations.

TdT-dUTP Nick-end-labeling Assay

Epi 4 cells were grown in a Lab-Tek™ Chamber Slide at a density of 5 × 10⁴ cells/well. Apoptotic cells were detected by the terminal deoxynucleotidyl transferase (TdT)-mediated biotin-dUTP nick-end-labeling (TUNEL) method with the In situ Apoptosis Detection Kit (Takara Bio, Inc., Shiga, Japan). Briefly, the cells were fixed with 4% paraformaldehyde for 15 min, permeabilized for 5 min, incubated with TdT end-labeling cocktail for 60 min, and then incubated with anti-fluorescein isothiocyanate (FITC) conjugate for 30 min. To quantitate apoptotic cell death, we calculated the percentage of TUNEL-positive cells relative to total cells after counting the numbers of TUNEL-positive cells and total cells in three random fields using a fluorescent microscope.

cDNA Microarray Analysis

RNA samples were amplified and labeled with Cy3 and a Quick Amp Labeling Kit (Agilent Technologies, Inc., Santa Clara, CA, USA), according to the manufacturer’s protocol. Following labeling and purification, Cy3-labeled cRNAs were competitively hybridized onto an Agilent 4 × 44 K whole human genome oligo microarray slide (Agilent Technologies, Inc.), then scanned in an Agilent GeneArray Scanner (Agilent Technologies, Inc.). The detailed protocol and microarray data used in our study were deposited in the National Center for Biotechnology Information Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo; Accession Numbers: GSE57759).

Statistical Analysis

All experiments were performed in triplicate for each set of conditions and repeated at least twice. All data are expressed as the mean ± standard error of the mean. Statistical analyses were performed with GraphPad Prism (GraphPad Software, Inc., San Diego, CA, USA). The Student’s t test was applied to compare the differences between the groups. The means of multiple groups were compared by analysis of variance followed by Tukey’s test. A p value of < .05 was considered statistically significant.

Results

TRPV1 Expression and Localization in Gingival Tissue

First, we examined the mRNA expression profiles of TRPV family members by conventional PCR. All TRPV family members were detected in mouse and human gingival tissues, with the exception of Trpv5 (Figs. 1A, 1B). Of the TRPV channels, the physiological and biological roles of TRPV1 have been studied most extensively; therefore, we focused on identifying and characterizing TRPV1 in GECs in subsequent experiments. Trpv1 mRNA expression was detected by conventional PCR in the gingival tissues of five different individuals for both mice and humans (Figs. 1C, 1D). We further confirmed TRPV1 localization in gingival tissues by immunohistochemistry. TRPV1 immunoreactivity was predominantly found within the gingival epithelial layer (Fig. 1E). The suprabasal layers showed more intense immunostaining compared with the basal layers and the junctional epithelium (Fig. 1E).

Figure 1.

TRPV1 expression in the gingival epithelium. (A, B) PCR products of TRPV 1–6 after 33 cycles of amplification from mouse and human gingival tissues. Values show their predicted length in base pairs. Glyceraldehyde 3-phosphate dehydrogenase (Gapdh) was used as an internal control. Water samples were used as a negative control. (C, D) Expression of Trpv1 mRNA in five different individual samples from mouse and human gingival tissues, respectively. (E) Representative immunohistochemical staining of murine gingival sections with anti-TRPV1 antibody at low (left panel) and high (right 2 panels) magnification. TRPV1 immunoreactivity was detected in the basal and suprabasal layers (asterisk) and junctional epithelium (arrowhead). Nuclei were counterstained with hematoxylin. The lower panels are sections without primary antibody, which served as negative controls.

Expression of Functional TRPV1 in GECs

To confirm TRPV1 expression in GECs, we performed conventional PCR using homogeneous populations of GECs cultured in vitro. TRPV1 expression was evident in primary cultured human GECs, in the SV40-immortalized human gingival epithelial cell line, epi 4 (Fig. 2A), and in the murine gingival epithelial cell line, GE1 (Fig. 2B). To analyze TRPV1 protein expression, we first validated the specificity of the anti-TRPV1 antibody for HEK-293 cells overexpressing TRPV1, then confirmed TRPV1 expression in epi 4 cells by Western blotting (Fig. 2C). TRPV1 expression by GECs was also demonstrated by immunostaining with epi 4 cells (Fig. 2D). Functional TRPV1 expression was demonstrated by Ca²⁺ influx assay in GECs; epi 4 cells were stimulated with the TRPV1 agonist capsaicin (1 µM), causing significant intracellular Ca²⁺ increase (Fig. 2E). Analysis of these data, taken together, suggests that GECs express functional TRPV1 channels.

Figure 2.

Cultured gingival epithelial cells (GECs) express functional TRPV1. (A) Expression of Trpv1 mRNA was confirmed in primary GECs, in the human gingival epithelial cell line, epi 4, and (B) in the murine gingival epithelial cell line, GE1. E-cadherin (E-Cad) was used as an epithelial cell marker. Gapdh was used as an internal control. (C) Endogenous expression of TRPV1 protein in GECs, shown by Western blotting. Human embryonic kidney (HEK)-293 cells, transfected with the TRPV1 expression vector (HEK-TRPV1), served as a positive control. Untransfected cells (HEK) served as a negative control. Equal loading was assessed by GAPDH immunoblotting. (D) Epi 4 cells were grown in wells of Lab-Tek™ Chamber Slides to subconfluence (70%). After fixation, cells were incubated with anti-TRPV1 overnight at 4°C. Cells were counterstained with hematoxylin, and mounted for microscopy. The right panel is a section without primary antibody, which served as a negative control. (E) TRPV1-mediated intracellular Ca²⁺ influx in epi 4 cells. Intracellular Ca²⁺ changes were analyzed as fluorescence intensity changes of the green-fluorescent calcium indicator, Fluo-4. Capsaicin (0.1, 1 µM) treatment induced the elevation of intracellular [Ca²⁺] in a dose-dependent manner. A calcium ionophore (Ionomycin, 10 ng/mL) was used as a positive control. *p < .05 vs. unstimulated cells.

Epithelial TRPV1 Signaling Accelerates GEC Proliferation

Disruption of the epithelial barrier and the subsequent invasion of exogenous substances into the gingiva facilitate the progression of periodontal breakdown. Preservation of epithelial proliferation is crucial for maintenance of the epithelial barrier and protection of the host. We performed MTT and wound-healing assays to evaluate the role of TRPV1 signaling in GECs. A significant increase in the number of epi 4 cells was evident after treatment with capsaicin (1 µM) for 72 hr (Fig. 3A). Pretreatment of epi 4 cells with the TRPV1 antagonist (SB-366791) prevented this pro-proliferative effect, suggesting a TRPV1-dependent mechanism (Fig. 3B). Consistent with these findings, wound-healing assays showed TRPV1-dependent, pro-proliferative effects of capsaicin on GECs (Figs. 3C, 3D). To assess the proliferative activity of capsaicin in vivo, we performed BrdU pulse-chase experiments. The mice received BrdU intraperitoneally and were euthanized 72 hr later, and their gingival tissues were processed for histologic analysis. As expected, the number of BrdU-positive cells was higher in mice fed with capsaicin compared with those fed the normal diet (Fig. 3E). In addition, transcript levels of proliferating cell nuclear antigen (PCNA) were significantly higher in the capsaicin-treated group than in the normal-diet group (Fig. 3F). No effects were observed on GEC apoptosis by epithelial TRPV1 signaling (Appendix Fig. A-D). Collectively, these results demonstrate the pro-proliferative effects of capsaicin on GECs in vitro and in vivo.

Figure 3.

Epithelial TRPV1 signaling accelerates GEC proliferation. (A) The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to examine the proliferative activity of epi 4 cells after treatment with the TRPV1 agonist, capsaicin, for 24, 48, and 72 hr. The relative cell proliferation of epi 4 treated with 1 µM capsaicin (solid square) for 72 hr was significantly increased compared with that of untreated cells (open circle). (B) Pretreatment with the TRPV1 antagonist, SB-366791 (1 µM), for 30 min inhibited capsaicin-induced cell proliferation at 72 hr. SB-366791 treatment alone had no effects on the proliferation of GECs. (C) To further confirm the effect of TRPV1 on proliferation in vitro, we performed wound-healing assays with epi 4 cells. Representative images after capsaicin treatment (1 µM) with or without SB-366791 (1 µM) for 24 hr are shown. Dotted lines indicate the margins of cells. (D) The level of cell migration into the wound scratch was quantified as the percentage of wound healing in the untreated group after 24-hour treatment. (E) Representative bromodeoxyuridine (BrdU) staining of murine gingival sections. Mice were treated with dietary capsaicin (100 parts per million in chow) for 7 days before BrdU injection. Increased numbers of BrdU-positive cells (arrowheads) were found in the gingiva of the capsaicin-treated group compared with the normal-diet group. (F) Expression of a proliferation marker, proliferating cell nuclear antigen, was significantly higher in the capsaicin-treated group. *p < .05; **p < .01 vs. unstimulated cells or the normal-diet group.

Identification of Candidate Genes Associated with TRPV1-dependent GEC Proliferation

To elucidate the molecular mechanisms of capsaicin-induced GEC proliferation, we performed cDNA microarray analysis. Gene expression profiles of the epi 4 cells stimulated with capsaicin (1 µM) for 4 hr were compared with those of untreated cells (four samples/group). The data were normalized and filtered by a fold change of > 1.5 and < 1.5 with an adjusted p value < .05, respectively. The 227 up-regulated and 232 down-regulated genes were sorted according to Gene Ontology “growth factor activity” (GO: 0008083). In total, four genes were identified and validated in independent experiments by quantitative PCR. Both fibroblast growth factor (FGF)-17 and neuregulin (NRG) 2 were significantly up-regulated in capsaicin-treated epi 4 cells relative to untreated cells (Fig. 4). Analysis of these data suggests that TRPV1 signaling in GECs may induce transcriptional up-regulation of growth factors, resulting in increased proliferation.

Figure 4.

Expression of candidate genes related to TRPV1-mediated cell proliferation. Epi 4 cells were stimulated with 1 µM capsaicin for 4 hr, and the gene expression profiles were analyzed by microarray. Among the up-regulated genes found to be related to cell proliferation by microarray analysis, statistical validation for fibroblast growth factor (FGF)-17 and neuregulin (NRG) 2 was discovered by independent experiments using quantitative polymerase chain-reaction. *p < .05; **p < .01 vs. unstimulated cells.

Discussion

In this study, we demonstrated that TRPV1 channels are robustly expressed by human and mouse GECs. Furthermore, we found that epithelial TRPV1 signaling promotes GEC proliferation. Previous studies have identified functional TRPV1 expression in other epithelial cells, including bronchial (Yang et al., 2013), corneal (Pan et al., 2011), and mammary (Kang et al., 2003) epithelial cells. In the oral cavity, Wang et al. (2011) reported TRPV1 mRNA expression in three oral epithelia (buccal, palatal, and lingual) and also found that TRPV5 mRNA was not expressed in these regions. These expression profiles are consistent with our results. To the best of our knowledge, our study provides the first evidence of TRPV1 expression in human and mouse GECs. Analysis of our data also suggests that GECs act as sensors of changes in the physical and chemical environment and may function in protecting against noxious stimuli from exogenous substances by promoting GEC proliferation.

Although we have demonstrated that TRPV1 signaling increases GEC proliferation in vitro, previous studies on the role of TRPV1 in proliferation are conflicting. Involvement of TRPV1 in pro-proliferative effects was reported in prostate epithelial cells (Malagarie-Cazenave et al., 2009), colonic epithelial cells (Liu et al., 2012), and human keratinocytes (Denda et al., 2010). In contrast, anti-proliferative effects of TRPV1 were found in lung (Brown et al., 2010) and prostate stromal (Venier et al., 2012) cells. The effects elicited by capsaicin may depend on the cell or tissue types studied. In oral epithelia, a proliferation marker, PCNA, expression was predominantly expressed in the suprabasal layers in the oral gingival epithelium (Celenligil-Nazliel et al., 2003), suggesting that the proliferating cells of the gingival epithelium are mainly localized in the suprabasal rather than in the basal layers. Our immunohistochemical staining of periodontal tissues showed that TRPV1 immunoreactivity was most intense in the suprabasal layer, indicating that TRPV1 expressed in the gingival epithelium acts as an inducer of cell proliferation rather than an anti-proliferative effector.

To determine the molecular mechanism of TRPV1-mediated GEC proliferation, we performed cDNA microarray analysis. Up-regulation of two proliferation-related genes, FGF-17 and NRG2, in capsaicin-stimulated GECs was validated by quantitative PCR. Both FGF-17 and NGR2 are growth factors involved in a variety of biological processes, including cell growth (Falls, 2003; Polnaszek et al., 2004). In addition, various FGF family members have been demonstrated to contribute to the autocrine regulation of epithelial cell proliferation (Maheshwari et al., 2001). Takayama et al. (2002) reported the expression of cognate receptors for FGF on GECs. This suggests that TRPV1 activation contributes to periodontal tissue homeostasis by triggering a positive feedback loop involving the production of multiple growth factors.

Disruption of periodontal tissue homeostasis has the potential to cause the initiation and progression of periodontitis (Hajishengallis, 2014). One limitation of our study is its lack of information on the possible involvement of TRPV1 in the pathogenesis of periodontal diseases. A recent study using immunohistochemistry showed that TRPV1 expression and distribution differed in subgingival specimens from patients with periodontitis and healthy individuals (Öztürk and Yildiz, 2011). However, further studies using both immunohistochemical and other biological methods are required. A more thorough understanding of the mechanism of action of capsaicin in GEC biology could potentially lead to the development of new therapeutic approaches for periodontal diseases.

In conclusion, we confirmed the expression of TRPV1 by GECs isolated from humans and mice. Our results suggest that TRPV1 channels function in the detection of a variety of chemical stimuli and in modulating epithelial wound-healing by affecting proliferation. Further studies are required to elucidate the contribution of TRPV1 to periodontal diseases.