in vivo 35 : 2099-2106 (2021) doi:10.21873/invivo.12479 Role of S100A4 in the Pathogenesis of Human Periapical Granulomas TAKAHITO TAMURA 1,2 , TAIKI MIYATA 1,2 , KEISUKE HATORI 1,3 , KAZUMA HIMI 1, TAKESHI NAKAMURA 1,2 , YURIKA TOYAMA 1,2 and OSAMU TAKEICHI 1,3 1Department of Endodontics, Nihon University School of Dentistry, Tokyo, Japan; 2Nihon University Graduate School of Dentistry, Dental Research Center, Tokyo, Japan; 3Division of Advanced Dental Treatment, Dental Research Center, Tokyo, Japan Abstract. Background/Aim: S100A4 expression is periodontitis is to eliminate the source of root canal infection associated with the pathology of chronic inflammatory that causes lesions of apical periodontal tissue and has a high diseases. In this study, we investigated the role of S100A4 success rate (4). However, despite proper treatment by the and four inflammatory mediators (IL-1 β, IĸB, IL-10, and dentist, apical periodontitis may not heal and tooth extraction TNF- α) in human periapical granulomas (PGs). Materials may be required (4). Cytokines and growth factors play an and Methods: S100A4 expression in PGs obtained by important role in the development of periapical lesions and apicoectomy was examined by immunohistochemistry. associated tissue damage (5). Notably, these inflammatory Further, the expression of S100A4 and four inflammatory mediators increase lymphocyte activation, inflammatory cell mediators was compared between PGs and healthy gingival migration, and osteoclast activation in the lesions (3, 6-8). tissues (HGTs) using real-time PCR. Results: In the PGs, Although various studies have investigated the mechanisms S100A4 was found to be expressed in endothelial cells and underlying the development of periapical periodontitis (1-8), fibroblasts. Furthermore, real-time PCR revealed that the several details remain unclear. Thus, the elucidation of the expression of S100A4 and IL-1 β in PGs was significantly pathology of apical periodontitis will be very useful for higher than that in HGTs. Although a correlation between endodontic treatment. the expression of S100A4 and IĸB or IL-10 was not detected, Calcium-binding proteins S100 (termed S100 proteins) are a positive correlation between the expression of S100A4 and low-molecular-weight proteins harbouring an EF-hand-type IL-1 β or TNF- α was observed. Conclusion: The expression calcium-binding domain (9). The S100 protein family of S100A4 correlates with the pathogenesis of PGs. comprises 25 proteins and is expressed only in vertebrates (10, 11). A member of the S100 protein family is known to Periapical granulomas (PGs), a type of refractory periapical be secreted by tumour cells and stromal cells, where S100 periodontitis, arise in response to the presence of oral bacteria supports tumorigenesis by inducing angiogenesis (11, 12). and their products (1, 2). PGs are primarily characterised by S100 proteins exhibit a cell-specific expression pattern and chronic inflammation around the tooth apex, a phenomenon are involved in regulating processes, such as cell proliferation responsible for bone resorption and the manifestation of and differentiation, apoptosis, and inflammation (13). various clinical symptoms, resulting in a marked reduction in Furthermore, recent studies have reported S100 expression to the quality of life of the patients (3). Treatment of apical be associated with chronic inflammatory diseases such as rheumatoid arthritis and Crohn’s disease, and that these proteins play a role in determining disease pathology (12, 14- This article is freely accessible online. 17). A prior study has shown that an S100 protein, S100A4, is involved in metastasis and is a major player in the Correspondence to: Keisuke Hatori, DDS, Ph.D., Department of transition from benign growth to malignancy (18). Endodontics, Nihon University School of Dentistry, 1-8-13 Kanda- Additionally, it has been suggested that S100A4 plays an Surugadai, Chiyoda-ku, Tokyo 101-8310, Japan. Tel: +81 essential role in the induction of chronic inflammation (19). 332198142, Fax: +81 332198348, e-mail: hatori.keisuke@nihon- In autoimmune diseases, the expression of interleukin-1 β ( IL- u.ac.jp 1β), tumour necrosis factor- α ( TNF- α), and interleukin-10 (IL- Key Words: Apical periodontitis, endothelial cells, S100A4, 10) in mononuclear cells is associated with that of S100A4 (20- inflammatory mediators, periapical granulomas, real-time 23). The binding of S100A4 to the receptor for advanced polymerase chain reaction. glycation end products activates nuclear factor-kappaB (NF-ĸB) 2099 in vivo 35 : 2099-2106 (2021) Table I . PCR primers used to detect the mRNA levels of S100A4, IL-1 β, IĸB, IL-10, TNF- α, and GAPDH in periapical granulomas and healthy gingival tissues. Gene Primer sequence GenBank accession number S100A4 Forward 5’-GTGTCCACCTTCCACAAATACTCA-3’ NM_002961 Reverse 5’-ACTTCATTGTCCCTGTTGCTGTC-3’ IL-1 β Forward 5’-CCAGGGACAGGATAGGAGCA-3’ NM_000576 Reverse 5’-TTCAACACGCAGGACAGGTACAG-3’ IĸB Forward 5’-CCAGTTGCAGGTGGCCTATC-3’ NM_1099857 Reverse 5’-CTGCTGGAGCTGCTGTTTGAG-3’ IL-10 Forward 5’-GAGATGCCTTCAGCAGAGTGAAGA-3’ NM_000572 Reverse 5’-AGTTCACATGCGCCTTGATGTC-3’ TNF- α Forward 5’-CTGCCTGCTGCACTTTGGAG-3’ NM_000594 Reverse 5’-ACATGGGCTACAGGTTGTCACT-3’ GAPDH Forward 5’-GCACCGTCAAGGCTGAGAA-3’ NR_152150 Reverse 5’-ATGGTGGTGAAGACGCCAGT-3’ and mitogen-activated protein kinase pathways, thereby conducted in accordance with the tenets of the Declaration of promoting tumour cell invasion and survival (24). S100A4 also Helsinki. Periapical periodontitis samples were obtained from impairs osteoblast-mediated calcification, thereby inhibiting patients (n=44; 26 men and 18 women; age, 32-64 years) during endodontic surgery and tooth extraction upon clinical examination. new bone formation and causing an imbalance in bone No systemic disease was observed in the patients. Additionally, the homeostasis (25). Via activating NF-ĸB, S100A4 interferes with patients did not report consuming any medications (for six months the calcification function of osteoblasts, inhibits the formation prior to the endodontic surgery) that could affect the inflammatory of new bone, and causes an imbalance in bone homeostasis responses. HGTs were obtained when impacted wisdom teeth were (25). Inhibitor of ĸB (IĸB), bound to the NF-ĸB dimer, is extracted from patients at the Department of Oral Surgery (n=5; 3 phosphorylated and degraded by the IĸB kinase (IKK) complex; men and 2 women; age, 25-37 years). as a result, NF-ĸB is activated and translocates to the nucleus Preparation of tissue sections. The samples were divided in half (26-28). Additionally, S100A4 downregulation suppresses bone immediately after the periapical lesions were excised. One half of resorption in periodontal diseases (29-31). Hence, elucidating the divided tissues was fixed with 10% neutral-buffered formalin the pathophysiology of apical periodontitis may aid in the and paraffin sections (4- μm thick) were prepared. The other half of development of endodontic treatment for refractory periapical the tissue samples was fixed with 4% paraformaldehyde prepared periodontitis. in phosphate-buffered saline (PBS), embedded in OCT compound Based on the role of S100A4 in periodontal diseases, we (Tissue-Tek, Elkhart, IN, USA), and frozen in dry-ice acetone. hypothesised that S100A4 is expressed in inflamed periapical These samples were then used for RNA preparation. HGT samples were also prepared as described above. tissues in humans and may be involved in the pathogenesis of PGs. The aim of this study was to compare the expression Histological examination. Haematoxylin-eosin (HE) staining was of S100A4 between inflammatory and healthy tissues in the performed to investigate tissue histopathology. After staining, three oral cavity. The expression of S100A4 (at protein and mRNA different fields in each tissue section were examined under a light levels) in PGs and healthy gingival tissues (HGTs) was microscope (Olympus BX60, Tokyo, Japan). assessed using immunohistochemistry and real-time polymerase chain reaction (PCR). In addition, we Immunohistochemistry. Paraffin sections were subjected to immunohistochemistry to identify S100A4-expressing inflammatory investigated the correlation between the expression of S100 cells in human PGs. The paraffin sections were first treated for 30 and that of other inflammatory mediators ( IL-1 β, IĸB , IL-10 , min at room temperature (RT) with 0.3% H 2O2 prepared in and TNF- α) in PGs using real-time PCR. In the future, the methanol to inactivate endogenous peroxidase, and non-specific results of this study may be useful to elucidate the binding was blocked by incubation in 1.5% normal horse serum pathophysiology of periapical periodontitis. (Vector Laboratories, Burlingame, CA, USA) for 90 min. The sections were probed with a rabbit anti-human S100A4 monoclonal antibody (1:100; Abcam, Cambridge, UK) in a humid atmosphere Materials and Methods at RT for 2 h. Then, the sections were washed with PBS and incubated with biotinylated horse anti-rabbit IgG antibody (1:500; Sample collection. The Ethics Committee of the Nihon University Vector Laboratories) at RT for 30 min. This was followed by an School of Dentistry approved the experimental protocol of this incubation with the avidin-biotin peroxidase complex (Vector study (EP18D014), and all patients provided written informed Laboratories) for 30 min. Optimum colour development of positive consent for treatment and publication of findings. The study was cells was achieved by incubating