IL-1Β Enhances Cell Adhesion Through Laminin 5 and Β4 Integrin in Gingival

491 Journal of Oral Science, Vol. 61, No. 4, 491-497, 2019 Original IL-1β enhances cell adhesion through laminin 5 and β4 integrin in gingival epithelial cells Masaru Mezawa1,2), Yuto Tsuruya1), Mizuho Yamazaki-Takai1), Hideki Takai1,2), Yohei Nakayama1,2), Christopher A. McCulloch3), and Yorimasa Ogata1,2) 1)Department of Periodontology, Nihon University School of Dentistry at Matsudo, Matsudo, Japan 2)Research Institute of Oral Science, Nihon University School of Dentistry at Matsudo, Matsudo, Japan 3)Matrix Dynamics Group, Faculty of Dentistry, University of Toronto, Toronto, Canada (Received November 26, 2018; Accepted December 15, 2018) Abstract: The junctional epithelium and dental enamel adhere because of hemidesmosomes containing laminin 5 and α6β4 integrin, which Introduction are important adhesion molecules in the internal The gingival epithelium comprises the oral epithelium, basal lamina. Interleukin (IL)-1 is important in the sulcular epithelium, and junctional epithelium (JE). The pathogenesis of periodontal disease. IL-1β induces JE forms apical to the dento-epithelial junction to the bone resorption by activating osteoclasts; however, sulcus. The coronal end of the JE forms the bottom of the its effects on adhesion of epithelial cells remain to be gingival sulcus and overlaps with the sulcular epithelium clarified. Laminin β3, β4 integrin, and focal adhesion (1,2). The JE has two basal laminas—the internal basal kinase mRNA levels were higher after 1 h and 3 h of lamina faces the tooth and the external basal lamina faces stimulation with IL-1β (1 ng/mL), and IL-1β, type I the gingival connective tissue. Hemidesmosomes are α1, and type IV α1 collagen mRNA levels were higher involved in promoting adhesion of epithelial cells to the after 1 h and lower after 3 h of stimulation with IL-1β. underlying basement membrane in stratified and other After IL-1β stimulation, colocalization of laminin 5 complex epithelia (4,5). The basal lamina is composed and β4 integrin was increased after 1 h, colocaliza- of extracellular matrix containing laminin, proteoglycan, tion of β4 integrin and plectin was increased after 1 and type IV collagen. However, laminin 5 is found only h and decreased after 3 h, and colocalization of β4 in the internal basal lamina, which lacks type IV collagen, integrin and type IV collagen was decreased after 3 h. and both these proteins are present in the extracellular Wound healing assays showed that IL-1β treatment (3 matrix. Laminin 5 consists of α3, β3, and γ2 subunits and h) delayed wound healing. These results suggest that contributes to cell adhesion associated with α6β4 integrin IL-1β enhances cell adhesion by altering localization at hemidesmosomes. Laminin 5 and α6β4 integrin were of epithelial adhesion molecules. reported to be involved in wound healing of epithelial tissues (6-9). Plectin, a functionally and universal Keywords: basal lamina; cell adhesion; hemidesmosome; versatile cytolinker protein, has been implicated in inflammatory cytokine; junctional epithelium. hemidesmosome functions, as early binding of plectin to the β4 subunit of α6β4 integrin is a critical step for Correspondence to Dr. Yorimasa Ogata, Department of hemidesmosome formation, and regions of plectin were Periodontology, Nihon University School of Dentistry at Matsudo, shown to be involved in the interaction (10,11). 2-870-1 Sakaecho-nishi, Matsudo, Chiba 271-8587, Japan Interleukin (IL)-1β is an essential mediator of proin- Fax: +81-47-360-9362 E-mail: [email protected] flammatory response (12) and is involved in defense J-STAGE Advance Publication: September 21, 2019 Color figures can be viewed in the online issue at J-STAGE. against external challenges, such as microorganisms, doi.org/10.2334/josnusd.18-0434 environmental irritants, and injury, and in innate immu- DN/JST.JSTAGE/josnusd/18-0434 nity and periodontitis pathophysiology (13). In adherent 492 fibroblasts, IL-1β induces release of matrix metal- grin reverse, 5′-CGGGTAGTCCTGTGTCCTGTA-3′; loproteinases (MMPs), which remodel the extracellular laminin α3 chain forward, 5′-TGCTAACAG- matrix (14,15) and enhance tissue repair after short-term TATCCGGGATTCT-3′; laminin α3 chain reverse, perturbations of connective tissue homeostasis, such as 5′-TCTTGGTTCAAGCCATTTGCC-3′; laminin β3 acute injury (16-18). IL-1β has a central role in tissue chain forward, 5′-CCAAGCCTGAGACCTACTGC-3′; destruction in many chronic inflammatory diseases, such laminin β3 chain reverse, 5′-GAATCTCCTGTC- as rheumatoid arthritis, pulmonary and cardiac fibrosis, CAGGTCCA-′3; laminin γ2 chain forward, inflammatory bowel disease, type 2 diabetes, and peri- 5′-GACAAACTGGTAATGGATTCCGC-3′; laminin γ2 odontitis (19,20). In this study, we analyzed the effects of chain forward, 5′-TTCTCTGTGCCGGTAAAAGCC-3′; IL-1β on adhesion molecules in gingival epithelial cells. type IV α1 collagen forward, 5′-ACCTGGT- CAAACTGGTCCTG-3′; type IV α1 collagen reverse, Materials and Methods 5′-GTGTCCCCTAATGCCTTTGA-3′; type I α1 collagen Reagent forward, 5′-GCTTGGTCCACTTGCTTGAAGA-3′; Rabbit polyclonal antibodies (laminin 5 and type IV type I α1 collagen reverse, 5′-GAGCATTGCCTTT- collagen), mouse monoclonal antibodies (β4 integrin), GATTGCTG-3′; focal adhesion kinase (FAK) forward, Phalloidin-iFluor 488 reagent, goat anti-rabbit Alexa 5′-GCCGTCATCTTCACGGACAG-3′; FAK reverse, 488, and goat anti-mouse Alexa 647 were obtained from 5′-AGGGTGTGTGAAGCCGTCAG-3′; MMP3 Abcam (Tokyo, Japan). Rabbit polyclonal antibodies forward, 5′-CTGGGCCAGGGATTAATGGAG-3′; to plectin were purchased from Novus (Littleton, CO, MMP3 reverse, 5′-CAATTTCATGAGCAACGAGA-3′; USA). Human recombinant IL-1β and alpha-minimum tissue inhibitor of metalloproteinase-1 (TIMP1) forward, essential medium (αMEM) were purchased from Wako 5′-AAGACCTACACTGTTGGCTGTGAG-3′; TIMP1 (Tokyo, Japan). Fetal calf serum (FCS), penicillin and reverse, 5′-GTCCGTCCACAAGCAATGAG-3′; streptomycin, TrypLE Express, and TRIzol Reagent were glyceraldehyde-3-phosphate dehydrogenase (GAPDH) purchased from Invitrogen (Carlsbad, CA, USA). The forward, 5′-GCACCGTCAAGGCTGAGAAC-3′; and PrimeScript RT reagent kit and SYBR Premix Ex Taq II GAPDH reverse, 5′-ATGGTGGTGAGACGCCAGT-3′. were purchased from Takara Bio (Tokyo, Japan). Bovine The amplification reactions were performed in 25 μL of serum albumin (BSA), complete protease inhibitor cock- final volume containing ×2 SYBR Premix EX Taq (12.5 tail, and phenylmethylsulfonyl fluoride were purchased μL), 0.4 μm forward and reverse primers (0.2 μL), and from Sigma Aldrich Japan (Tokyo, Japan). Anti-mouse 70 ng cDNA (7 μL) for β4 integrin, laminin α3, laminin IgG (whole molecule) peroxidase antibody produced in β3, laminin γ2, type IV α1 collagen, type I α1 collagen, rabbit, anti-rabbit IgG (whole molecule) peroxidase anti- FAK, MMP3, and TIMP1, and 50 ng cDNA (5 μL) for body produced in goat, and ECL Prime Western Blotting GAPDH. To reduce variability between replicates, PCR Detection Reagents were purchased from GE Healthcare premixes containing all reagents except for cDNA were (Buckinghamshire, UK). All chemicals used were of prepared and aliquoted into 0.2-mL PCR tubes (Nippon analytical grade. Genetics). The thermal cycling conditions were 10 s at 95°C, 45 cycles of 5 s at 95°C, and 30 s at 60°C. Post-PCR Cell cultures melting curves confirmed the specificity of single-target Human gingival epithelial Ca9-22 cells were cultured in amplification, and expressions of β4 integrin, laminin α3, αMEM containing 10% FCS until 70-80% confluent in laminin β3, laminin γ2, type IV α1 collagen, type I α1 5% CO2 and 95% air at 37°C. Ca9-22 cells were stimu- collagen, FAK, MMP3, and TIMP1 relative to GAPDH lated with IL-1β (1 ng/mL) for 1 h and 3 h. were determined in triplicate. Real-time PCR Wound healing assay Total RNA was isolated by TRIzol Reagent according Ca9-22 cells were cultured in αMEM containing 10% to the manufacturer’s protocol. Total RNA (1 μg) was FCS on fibronectin (10 μg/mL)-coated 35-mm culture used as a template for cDNA, which was prepared with dishes and grown to confluence. After the medium was the PrimeScript RT reagent kit. Using SYBR Premix changed to αMEM containing 1% FCS, cell layers were Ex Taq II in a TP800 Thermal Cycler Dice Real-Time scratched at a diameter of about 3 mm with a 1-mm- System (Takara Bio), we performed quantitative real- wide cell scraper (Corning) in the center of the dishes time PCR with the following primer sets: β4 integrin and washed twice with phosphate-buffered saline (PBS) forward, 5′-CTCCACCGAGTCAGCCTTC-3′; β4 inte- to remove the detached cells. The cells were stimulated 493 with IL-1β (1 ng/mL) for 1 h and 3 h and then cultured Statistical analysis for 6 days to analyze the wound healing area. In all experiments, separate assays were repeated at least three times. For quantitative data, means ± SEM Western blot was computed. Comparisons of multiple samples were Ca9-22 cells were lysed, and equal amounts of protein analyzed with ANOVA. Statistical significance was set were loaded and separated on 8% SDS-PAGE gels and at P < 0.05. transferred to Hybond 0.2-μm polyvinylidene fluoride membranes. The membranes were blocked and probed Results with the various primary antibodies at 4°C overnight, Regulation of adhesion molecule mRNA levels by followed by fluorescent secondary antibody incuba- IL-1β tion for 1 h at room temperature. The membrane was To determine whether IL-β affects mRNA levels of incubated with anti-β4 integrin (ab29042; Abcam, cell adhesion molecules in Ca9-22 cells, we stimulated Cambridge, UK), anti-laminin 5 (ab14509; Abcam), type Ca9-22 cells with IL-1β (1 ng/mL) for 1 h and 3 h. β4 IV collagen (ab6586; Abcam), and anti-αtubulin (sc5286; integrin, laminin β3, and FAK mRNA levels increased Santa Cruz Biotechnology, CA, USA) antibodies for 2 h. after 1 h and 3 h of IL-1β stimulation (Fig. 1A-C) Anti-rabbit and anti-mouse IgG conjugated with horse- Laminin α3 and laminin γ2 mRNA levels increased after radish peroxidase were used as the secondary antibodies.

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