Expression of SARS-CoV-2 entry factors in human oral tissue.

The distribution of cells expressing SARS-CoV-2 entry factor angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2) in human oral tissues were tested. The investigation was conducted with normal flesh tissue and paraffin-embedded specimens. The ACE2 and TMPRSS2 expression was detected with all subjects in the normal mucosa of the keratinized stratified squamous epithelia of the tongue and non-keratinized stratified squamous epithelia of the lip and cheek. It was found that ACE2 is expressed in the cytoplasm and on the cell membrane mainly in the stratum granulosum of the epithelia while the TMPRSS2 is strongly expressed on the cell membrane mainly in the stratum granulosum and stratum spinosum, but not in the stratum basale. Antibodies' reactions for ACE2 and TMPRSS2 were not observed in the nuclei or keratin layer. The expression of ACE2 and TMPRSS2 in the oral epithelia appears to be general, and the expression was also observed in the mucous and serous acini of the labial glands. The SARS-CoV-2 may transiently attach to the oral mucosa and the minor salivary glands which are present under all of the oral mucosa. The oral cavity can be considered an important organ for SARS-CoV-2 attachment and may provide a preventive medical avenue to guard against COVID-19 by preventing saliva from scattering.

Cisplatin-induced programmed cell death ligand-2 expression is associated with metastasis ability in oral squamous cell carcinoma.

Programmed cell death ligands (PD-Ls) are expressed in tumor cells where they bind to programmed cell death-1, an immunocyte co-receptor, resulting in tumor cell evasion from the immune system. Chemotherapeutic drugs have been recently reported to induce the expression of PD-L, such as PD-L1, in some cancer cells. However, little is known regarding PD-L2 expression and its role in oral squamous cell carcinoma (OSCC). In this study, we examined the effect of cisplatin on the expression and regulation of PD-L2 in OSCC cell lines and analyzed malignant behavior in PD-L2-expressing cells using colony, transwell and transformation assays. In addition, we examined PD-L2 expression in the tumor tissues of OSCC patients using cytology and tissue microarray methods. In OSCC cell lines, cisplatin treatment upregulated PD-L2 expression, along with that of the drug efflux transporter ABCG2, via signal transducers and activator of transcription (STAT) 1/3 activation. Moreover, PD-L2-positive or PD-L2-overexpressing cells demonstrated upregulation in both invasion and transformation ability but not in proliferation compared with PD-L2-negative or PD-L2-silencing cells. PD-L2 expression was also observed in OSCC cells of cytology samples and tissue from OSCC patients. The intensity of PD-L2 expression was correlated with more malignant morphological features in the histological appearance and an invasive pattern. Our findings indicate that cisplatin-upregulated PD-L2 expression in OSCC via STAT1/3 activation and the expression of PD-L2 are likely to be associated with malignancy in OSCC. The PD-L2 expression in cisplatin-resistant OSCC cells may be a critical factor in prognosis of advanced OSCC patients.

Mild Heat Stress Affects on the Cell Wall Structure in Candida albicans Biofilm.

We previously reported that Candida albicans responded to mild heat stress in a range of temperature elevations simulating fever, and concluded that mild heat stress increases susceptibility to antifungal drugs. In this study, we show that mild heat stress causes a morphological change in hyphae during the process of biofilm formation. We found that mild heat stress extended the period of hyphal stage maintenance in C. albicans biofilm. Although the rate of hyphal change from yeast form to hyphal form reached the maximum within 3 hr, later, almost every cell quickly reverted to the yeast growth phase within 6 hr at 37°C but not at 39°C, or under mild heat stress. Electron microscopy using a smart specimen preparation technique revealed that mild heat stress significantly increased the thickness of the inner cell wall accompanied by a decrease in density of the outer cell wall in the hyphae of C. albicans biofilm. To identify the gene responsible for the morphological changes associated with mild heat stress, we performed microarray gene expression analysis. Eleven genes were upregulated and 17 genes were downregulated under mild heat stress in biofilm cells. The increased PHR1 gene expression in response to mild heat stress was confirmed in quantitative RT-PCR analysis. The mutant upregulated PHR1 expression showed the same sensitivity against antifungal drug micafungin as dependent on mild heat stress. Our findings point to possible therapeutic effects of hyperthermia as well as to the effect of fever during infections.

Nuclear PKM2 promotes the progression of oral squamous cell carcinoma by inducing EMT and post-translationally repressing TGIF2.

Pyruvate kinase M2 (PKM2), a glycolytic enzyme, acts as a metabolic function leading to an energy production critical for cancer progression, known as Warburg effect. In this study we showed a pivotal role of PKM2 acting as a non-metabolic function to promote cancer cell progression in human oral squamous cell carcinoma (OSCC) through the induction of epithelial-mesenchymal transition (EMT), which is crucial for the potential in cancer cell invasion, and post-translational TGIF2 degradation. PKM2 immunoreaction was strong in the cytoplasm of invasive cancer cells, and distinct in the nucleus of spindle-shaped cancer cells with EMT characteristics. TGIF2 nuclear immunoreaction was seen in dysplastic epithelial cells but was repressed in cancer cells. In vitro analyses, cytoplasmic expression of PKM2 was translocated into the nucleus in human OSCC derived HSC-4 and SAS cells when EMT was stimulated. In addition, nuclear expression of TGIF2 was distinctively repressed in EMT induced HSC-4 and SAS cells. We recognized a mismatch in TGIF2 protein and mRNA expression in EMT induced HSC-4 and SAS cells and found that TGIF2 protein was post-translationally degraded through a ubiquitin proteasome system by an MG132 proteasome inhibition assay. Finally, promotion of HSC-4 and SAS cell progression by PKM2 was recognized in PKM2 knockdown assays. Thus, we clarified a new mechanism of non-metabolic function of PKM2 to promote the progression of OSCC through PKM2 nuclear translocation, subsequently induced EMT, and post-translationally repressed TGIF2 expression by a ubiquitin proteasome system.

Nutrient-induced FNIP degradation by SCFß-TRCP regulates FLCN complex localization and promotes renal cancer progression.

Folliculin-interacting protein 1 and 2 (FNIP1 and FNIP2) play critical roles in preventing renal malignancy through their association with the tumor suppressor FLCN. Mutations in FLCN are associated with Birt-Hogg-Dubé (BHD) syndrome, a rare disorder with increased risk of renal cancer. Recent studies indicated that FNIP1/FNIP2 double knockout mice display enlarged polycystic kidneys and renal carcinoma, which phenocopies FLCN knockout mice, suggesting that these two proteins function together to suppress renal cancer. However, the molecular mechanism functionally linking FNIP1/FNIP2 and FLCN remains largely elusive. Here, we demonstrated that FNIP2 protein is unstable and subjected to proteasome-dependent degradation via ß-TRCP and Casein Kinase 1 (CK1)-directed ubiquitination in a nutrition-dependent manner. Degradation of FNIP2 leads to lysosomal dissociation of FLCN and subsequent lysosomal association of mTOR, which in turn promotes the proliferation of renal cancer cells. These results indicate that SCFß-TRCP negatively regulates the FLCN complex by promoting FNIP degradation and provide molecular insight into the pathogenesis of BHD-associated renal cancer.

A salmon DNA scaffold promotes osteogenesis through activation of sodium-dependent phosphate cotransporters.

We previously reported the promotion of bone regeneration in calvarial defects of both normal and ovariectomy-induced osteoporotic rats, with the use of biodegradable DNA/protamine scaffold. However, the method by which this DNA-containing scaffold promotes bone formation is still not understood. We hypothesize that the salmon DNA, from which this scaffold is derived, has an osteoinductive effect on pre-osteoblasts and osteoblasts. We examined the effects of salmon DNA on osteoblastic differentiation and calcification in MC3T3-E1 cells, mouse osteoblasts, in vitro and bone regeneration in a calvarial defect model of aged mouse in vivo. The salmon DNA fragments (300 bps) upregulated the expression of the osteogenic markers, such as alkaline phosphatase, Runx2, and osterix (Osx) in MC3T3E1 cells compared with incubation with osteogenic induction medium alone. Measurement of phosphate ion concentrations in cultures showed that the DNA scaffold degraded phosphate ions were released to the cell cultures. Interestingly, we found that the inclusion of DNA in osteoblastic cell cultures upregulated the expression of sodium-dependent phosphate (NaPi) cotransporters, SLC20A1 and SLC34A2, in MC3T3-E1 cells in a time dependent manner. Furthermore, the inclusion of DNA in cell cultures increased the transcellular permeability of phosphate. Conversely, the incubation of phosphonoformic acid, an inhibitor of NaPi cotransporters, attenuated the DNA-induced expression and activation of SLC20A1 and SLC34A2 in MC3T3-E1 cells, resulting in suppression of the osteogenic markers. The implantation of a salmon DNA scaffold disk promoted bone regeneration using calvarial defect models in 30-week-old mice. Our results indicate that the phosphate released from salmon DNA upregulated the expression and activation of NaPi cotransporters, resulting in the promotion of bone regeneration.

Epigenetic alterations of the keratin 13 gene in oral squamous cell carcinoma.

BACKGROUND: Epigenetic modifications play important roles in the regulation of gene expression determining cellular phenotype as well as various pathologies such as cancer. Although the loss of keratin 13 (KRT13) is reportedly linked to malignant transformation of oral epithelial cells, the molecular mechanisms through which KRT13 is repressed in oral squamous cell carcinoma (OSCC) remain unclear. The aim of this study is to identify the epigenetic alterations of the KRT13 gene in OSCCs. METHODS: We investigated KRT13 expression levels and chromatin modifications of the KRT13 promoter in the three OSCC cell lines (HSC4, HSC3, and SAS). The expression levels of KRT13 protein and mRNA were analyzed by western blotting and quantitative reverse-transcription polymerase chain reaction, respectively, and the localization of KRT13 protein was detected by immunofluorescence. DNA methylation and histone modifications in the KRT13 promoter were determined by bisulfite sequencing and chromatin immunoprecipitation (ChIP), respectively. For the pharmacological depletion of Polycomb repressive complex 2 (PRC2), cells were treated with 3-deazaneplanocin A (DZNep). RESULTS: KRT13 expression was transcriptionally silenced in the HSC3 and SAS cells and post-transcriptionally repressed in the HSC4 cells, while the KRT13 promoter was hypermethylated in all of the three OSCC cell lines. ChIP analysis revealed that PRC2-mediated trimethylation of Lys 27 on histone H3 (H3K27me3) was increased in the KRT13 promoter in the HSC3 and SAS cells. Finally, we demonstrated that the treatment of SAS cells with DZNep reactivated the transcription of KRT13 gene. CONCLUSIONS: Our data provide mechanistic insights into the epigenetic silencing of KRT13 genes in OSCC cells and might be useful for the development of diagnostic markers and novel therapeutic approaches against OSCCs.

Reactive oxygen species promotes cellular senescence in normal human epidermal keratinocytes through epigenetic regulation of p16(INK4a.).

Reactive oxygen species (ROS) can cause severe damage to DNA, proteins and lipids in normal cells, contributing to carcinogenesis and various pathological conditions. While cellular senescence arrests the early phase of cell cycle without any detectable telomere loss or dysfunction. ROS is reported to contribute to induction of cellular senescence, as evidence by its premature onset upon treatment with antioxidants or inhibitors of cellular oxidant scavengers. Although cellular senescence is known to be implicated in tumor suppression, it remains unknown whether ROS initially contributed to be cellular senescence in normal human epidermal keratinocytes (NHEK) and their malignant counterparts. To clarify whether ROS induce cellular senescence in NHEKs, we examined the effect of hydrogen peroxide (H2O2) on the expression of cellular senescence-associated molecules in NHEKs, compared to in squamous carcinoma cells (SCCs). Hydrogen peroxide increased the number of cells positive in senescence associated-ß-galactosidase (SA-ß-Gal) activity in NHEKs, but not SCCs. The expression of cyclin-dependent kinase (CDK) inhibitors, especially p16(INK4a) was upregulated in NHEKs treated with H2O2. Interestingly, H2O2 suppressed the methylation of p16(INK4a), promoter region in NHEKs, but not in SCCs. Hydrogen peroxide also suppressed the expression of phosphorylated Rb and CDK4, resulting in arrest in G0/G1 phase in NHEKs, but not SCCs. Our results indicate that the ROS-induced cellular senescence in NHEKs was caused by the upregulation p16(INK4a) through demethylation in its promoter region, which is not detected in SCCs, suggesting that ROS-induced cellular senescence contributes to tumor suppression of NHEKs.

Inhibition of extracellular signal-regulated kinase downregulates claudin-2 expression and alters paracellular permeability in mouse rectum CMT93-II cells.

The morphological and physiological properties of tight junctions (TJs) are determined by the combination and mixing ratios of claudin species. Mouse rectum carcinoma cell lines, CMT93-I and -II cells, expressed claudin-4, -6, -7, and -12, and CMT93-II cells further expressed claudin-2. Although there were no differences in the morphology and number of TJ strands between the two cell lines, transepithelial electrical resistance (TER) of CMT93-II cells was approximately one-seventh that of CMT93-I cells. In this study, we aimed to determine whether claudin-2 expression in CMT93-II cells caused the reduction of TER. Inhibition of the extracellular signal-regulated kinase (ERK) pathway by U0126 treatment for 24 and 48h in CMT93-II cells markedly decreased claudin-2 from the apical junctional region and increased TER. However, claudin-4, -6, and -7 were still continuously localized at the apical junctional region by U0126 treatment. Moreover, the claudin-2 expression recovered at the apical junctional region after the removal of U0126 and TER decreased almost to the baseline level. These results suggest that the ERK pathway positively regulates claudin-2 protein expression and claudin-2 is involved in lowering TER in CMT93-II cells.

Reactive oxygen species stimulates epithelial mesenchymal transition in normal human epidermal keratinocytes via TGF-beta secretion.

Epithelial to mesenchymal transition (EMT) plays an important role in tumor progression, and is an early step in carcinogenesis. Although reactive oxygen species (ROS) are known to be implicated in EMT in many tumor cell types, its exact role in EMT initiation in normal human cells, especially epidermal keratinocytes (NHEKs), remains unknown. To clarify whether ROS induce EMT in NHEKs, and to establish how ROS regulate EMT, we examined the effect of hydrogen peroxide (H(2)O(2)) on the expression of molecules involved in EMT and cell morphology in NHEKs. H(2)O(2) altered the expression of EMT biomarkers, including downregulation of epithelial cadherin and upregulation of α-smooth muscle actin, through a transcriptional modulator, Snail1. H(2)O(2) also induced epithelial to fibroblast-like morphological changes, together with upregulation of EMT biomarkers, and promoted phosphorylation of ERK1/2 and JNK in a time-dependent manner. Interestingly, H(2)O(2) stimulated the expression and secretion of TGF-ß1 in NHEKs. Exogenous TGF-ß1 also induced the expression of EMT biomarkers. In contrast, neutralizing antibody anti-TGF-ß1 antibody or inhibitor of TGF-ß receptor type I suppressed the expression of EMT biomarkers. Our results suggest that ROS stimulated TGF-ß1 secretion and MAPK activation, resulting in EMT initiation in NHEKs.

Blockade of TRPM8 activity reduces the invasion potential of oral squamous carcinoma cell lines.

Several members of the transient receptor potential (TRP)-channel family are expressed in cancer cells. One, cold/menthol-sensitive TRPM8, is reportedly an important player in carcinogenesis in human prostate cancer, although its involvement in oral squamous cell carcinoma (SCC) remains unclear. The present immunohistochemistry and RT-PCR results revealed intense TRPM8 expression in two SCC cell lines, HSC3 and HSC4, derived from the human tongue. Menthol, icilin, and a more specific TRPM8 agonist (WS-12) induced non-specific cation currents, with Ca2+ permeability being greater than that of Na+ or K+. The novel TRPM8 antagonist RQ-00203078 (RQ) profoundly reduced such agonist-induced cation currents. Intracellular Ca2+ imaging revealed that menthol induced both intracellular Ca2+ release and store-operated Ca2+ entry, with RQ inhibiting each effect. To assess the possible pathophysiological role of TRPM8 in oral SCC, we performed motility and invasion assays, and gelatin zymography. Menthol augmented the migration and invasion abilities of both HSC3 and HSC4 cells by potentiating MMP-9 activity. RQ suppressed all of these effects. These results may aid understanding of the pathophysiological implications of TRPM8 channels in the oral SCC cells, support TRP proteins as valuable targets for pharmaceutical intervention, and inform the targeting of oral SCC in which the prognosis is poor.

Selective inhibition of nuclear factor-κB by nuclear factor-κB essential modulator-binding domain peptide suppresses the metastasis of highly metastatic oral squamous cell carcinoma.

Nuclear factor-κB (NF-κB) activation contributes to the development of metastasis, thus leading to a poor prognosis in many cancers, including OSCC. However, little in vivo experimental data are available about the effects of NF-κB inhibition on OSCC metastasis. OSCC sublines were established from a GFP-expressing parental cell line, GSAS, and designated GSAS/N3 and N5 according to the in vivo passage number after cervical lymph node metastasis by a serial orthotopic transplantation model. In vitro migration and invasion were assessed in these cells, and the NF-κB activities and expression of NF-κB-regulated metastasis-related molecules were also examined. In in vivo experiments, the metastasis and survival of tumor-engrafted mice were monitored. Furthermore, the effects of a selective NF-κB inhibitor, NEMO-binding domain (NBD) peptide, on metastasis in GSAS/N5-engrafted mice were assessed, and engrafted tongue tumors were immunohistochemically examined. Highly metastatic GSAS/N3 and N5 cells showed an enhanced NF-κB activity, thus contributing to increased migration, invasion, and a poor prognosis compared with the parent cells. Furthermore, the expression levels of NF-κB-regulated metastasis-related molecules, such as fibronectin, ß1 integrin, MMP-1, -2, -9, and -14, and VEGF-C, were upregulated in the highly metastatic cells. The NBD peptide suppressed metastasis and tongue tumor growth in GSAS/N5-inoculated mice, and was accompanied by the downregulation of the NF-κB-regulated metastasis-related molecules in engrafted tongue tumors. Our results suggest that the selective inhibition of NF-κB activation by NBD peptide may provide an effective approach for the treatment of highly metastatic OSCC.

Nationwide survey for bisphosphonate-related osteonecrosis of the jaws in Japan.

PURPOSE: A nationwide retrospective cohort study was conducted by the Japanese Society of Oral and Maxillofacial Surgeons to assess the occurrence of bisphosphonate (BP)-related osteonecrosis of the jaws (BRONJ) during 2006 to 2008 and to elucidate the outcome and factors associated with remission of BRONJ. MATERIALS AND METHODS: A written questionnaire, including the clinical characteristics, management, and outcome of patients with BRONJ, was sent to 248 institutions certified as training facilities by the Japanese Society of Oral and Maxillofacial Surgeons in 2008. RESULTS: A total of 568 patients with BRONJ, including suspicious cases, were registered. Of these 568 patients, 263, including the maxilla in 81, the mandible in 160, and both in 22, met the working definition of BRONJ proposed by the American Association of Oral and Maxillofacial Surgeons. The patients included 219 women (83.3%) and 44 men (16.7%). Of these patients, 152 (57.8%) had received intravenous BPs, 104 (39.5%) had received oral BPs, and 7 (2.7%) had received both. The mean duration of administration until onset of BRONJ was 23.6 months for intravenous BPs and 33.2 months for oral BPs. BRONJ was stage 1 in 42 patients (16.0%), stage 2 in 187 (71.1%), stage 3 in 32 (12.2%), and unknown in 2. Of these patients, 34.2% had remission of BRONJ, 46.0% had persistent or progressive disease, and 19.7% died of malignancy or were lost to follow-up. Statistical analysis revealed that surgical treatment, including tooth extraction, sequestrectomy, and segmental mandibulectomy, contributed to the remission of BRONJ. In contrast, conservative treatment, concurrent anticancer drugs, poor oral hygiene, and the use of intravenous BPs did not. CONCLUSIONS: The relative ratio of BRONJ related to the use of oral BPs was greater in Japan than in the United States and European Union. Surgical treatment contributed to remission of BRONJ, and conservative treatment, concurrent anticancer drugs, poor oral hygiene, and intravenous BPs did not.

Zoledronic acid inhibits RANK expression and migration of osteoclast precursors during osteoclastogenesis.

Bisphosphonates have been known to directly inhibit bone resorption and promote apoptosis in mature osteoclasts. Although bisphosphonates have been recognized as the most effective drugs to treat osteoporosis and bone cancer metastasis, the exact effects and mechanism(s) of bisphosphonates on osteoclastogenesis are unclear. The aim of this study was to clarify whether nitrogen-containing bisphosphonates affect recruitment and differentiation in osteoclasts. We examined the effects of zoledronic acid on receptor activator of NF-κB (RANK) expression and cell migration during osteoclastogenesis in two types of osteoclast precursors, RAW264.7 cells and Bone marrow cells (BMCs). Tumor necrosis factor-α (TNF-α) and RANK ligand (RANKL) upregulated RANK expression in RAW264.7 and BMCs in the presence of macrophage colony stimulating factor in a time-dependent manner. Zoledronic acid (30 and 50 µM) had no effect on cell viability in osteoclast precursors after 36 h of cultivation. Zoledronic acid (10 and 30 µM) strongly inhibited TNF-α- and RANKL-induced upregulation of RANK in a dose-dependent manner. The inhibitory effects on RANK expression were likely to be associated with the suppression of the NF-κB pathway, but not other downstream signaling pathways. Zoledronic acid (30 µM) also suppressed the TNF-α- and RANKL-induced migration of precursors by inhibiting the mevalonic acid pathway. Our results suggest that nitrogen-containing bisphosphonates not only inhibit mature osteoclasts but also prevent osteoclast precursors from differentiating and migrating towards inflammatory osteolysis lesions.

Assessment of the relationship between impacted mandibular third molars and inferior alveolar nerve with dental 3-dimensional computed tomography.

PURPOSE: The purpose of this study was to assess the capacity of dental 3-dimensional computed tomography (3D-CT; limited cone-beam CT) to predict the exposure and injury of the inferior alveolar nerve (IAN) after mandibular third molar extractions. MATERIALS AND METHODS: This study was a retrospective case series of patients who presented for extraction of mandibular third molars. Subjects eligible for study enrollment were those who underwent preoperative dental 3D-CT because the mandibular third molars were determined to be extremely close to the IAN on panoramic radiogram. The predictive variable was the anatomic relation of the IAN and third molar apices and was a binary variable, contact or noncontact. The primary outcome variable was IAN exposure, and the secondary outcome variable was IAN injury. RESULTS: From January 2006 to August 2007, 1,853 mandibular third molars in 1,539 patients were extracted. Among them, dental 3D-CT was performed on 53 third molars in 47 patients. The mandibular third molars were judged to make contact with the mandibular canal on dental 3D-CT images in 35 cases (66%). Intraoperative IAN exposure was observed in 17 (49%) contact cases and 2 (11%) noncontact cases on dental 3D-CT images. Of 53 cases extracted after dental 3D-CT examinations, IAN injury occurred in 8 cases (15%). IAN exposure led to IAN injury in 36.8% of cases, whereas IAN injury occurred in only 2.9% of cases without IAN exposure. Although the incidence of IAN injury in the molar-canal contact cases was 23%, all 8 cases with IAN injury (100%) were included in these contact cases. CONCLUSION: When viewing the anatomic relation between the IAN and mandibular third molar root apices using dental 3D-CT, contact of the 2 anatomic structures results in an increased risk for IAN exposure or injury.

Runt-related gene 2 is involved in the inhibition of matrix metalloproteinase-13 expression by roxithromycin in human gingival epithelial cell cultures.

BACKGROUND AND OBJECTIVE: Matrix metalloproteinase (MMP)-13 has wide substrate specificity compared with other MMPs and appears to be involved in periodontitis. Previously, we reported that roxithromycin (RXM) inhibits vascular endothelial growth factor expression induced by tumour necrosis factor-alpha in human periodontal ligament cells, but little is known about the effect of RXM on MMP-13 expression in human gingival epithelial cells. We therefore examined the effect of RXM on MMP-13 mRNA expression and production in cultured human gingival epithelial cells. MATERIAL AND METHODS: Human epithelial cell lines (Ca9-22, TU4, SCCTF and HSC-3) were plated in tissue culture dishes. Then, the culture supernatants and sediments were collected and the production of MMP-13 was analysed using enzyme-linked immunosorbent assay; the expression of MMP-13 mRNA and runt-related gene 2 mRNA was assessed using reverse transcriptase-polymerase chain reaction (RT-PCR) and real-time RT-PCR. We also studied the effect of Runx2 short interfering RNA (siRNA) on the induction of MMP-13. RESULTS: Roxithromycin downregulated the induction of MMP-13 in Ca9-22 cells. Roxithromycin suppressed the expression of MMP-13 mRNA not only in Ca9-22 cells, but also in other human epithelial cell lines. Roxithromycin strongly inhibited the expression of Runx2 mRNA. Furthermore, Runx2 siRNA inhibited the induction of MMP-13 in Ca9-22 cells. CONCLUSION: These results indicate that RXM suppresses MMP-13 via the downregulation of Runx2 in human gingival epithelial cell cultures.