Expression of human caspase-4 in the gingival epithelium affected with periodontitis: Its involvement in Porphyromonas gingivalis-challenged gingival epithelial cells.

OBJECTIVE: Implication of human caspase-4 in periodontitis and in sensing periodontal pathogens by gingival epithelial cells (GECs) is unclear. This study aimed to determine caspase-4 and interleukin (IL)-18 expressions in gingival tissues affected with periodontitis and to investigate caspase-4 involvement in mediating innate immune responses in GECs. DESIGN: Ex vivo, caspase-4 and IL-18 expressions in gingival biopsies, obtained from healthy participants with periodontitis or clinically healthy gingiva (N = 20 each), were determined by immunohistochemistry. In vitro, caspase-4 activation in cultured GECs stimulated with Porphyromonas gingivalis or Fusobacterium nucleatum was analyzed by immunoblotting. mRNA expressions of human ß-defensin-2 (hBD-2), IL-8, and IL-18 in stimulated GECs in the presence or absence of a caspase-4 inhibitor were assayed by RT-qPCR. RESULTS: Ex vivo, compared with healthy gingival epithelium, the epithelium affected with periodontitis displayed a significant decrease in caspase-4 expression (P = 0.015), whereas IL-18 expression was significantly increased (P = 0.012). Moreover, the expression of caspase-4, but not IL-18, was found to be a predictor of periodontitis (P = 0.007). In vitro, caspase-4 was activated in cultured GECs challenged with P. gingivalis, but not F. nucleatum. mRNA upregulations of hBD-2, IL-8, and IL-18 upon P. gingivalis stimulation were significantly reduced when caspase-4 was inhibited (P < 0.05), whereas the inhibitor failed to suppress those inductions by F. nucleatum. CONCLUSIONS: Caspase-4 expression is diminished in the epithelium affected with periodontitis while that of IL-18 is enhanced. Caspase-4 activation in P. gingivalis-infected GECs upregulates the three innate immune effector molecules, suggesting a possible sensing mechanism of caspase-4 in GECs in periodontal disease pathogenesis.

Overexpression of methyltransferase-like 3 and 14 in oral squamous cell carcinoma.

OBJECTIVES: This study aimed to determine expressions of methyltransferase-like 3 (METTL3) and METTL14, two enzymes essential for mRNA methylation at the adenosine (m6 A), in oral squamous cell carcinoma (OSCC) and to investigate in vitro aggressiveness of their aberrant expressions. METHODS: METTL3 and METTL14 expressions in 50 OSCC and 11 normal oral tissues were examined by immunohistochemistry. METTL3 and METTL14 expressions and m6 A amounts were determined in three OSCC cell lines, including HN5, HN6, and HN15. Cell proliferation, migration, and invasion were studied by BrdU, wound healing, and Transwell chamber assays, after silencing of METTL3, METTL14, or both by siRNA transfection. RESULTS: Immunostaining of METTL3 and METTL14 was localized in cancer cell nuclei. The mean percentages of METTL3- and METTL14-positive cells were significantly increased in OSCC tissues (p < 0.001). The percentages of METTL3- and METTL14-positive cells were correlated with the advanced pTNM stages (p < 0.05) and with the degrees of histopathological differentiation in OSCC (r = 0.564 and r = 0.316, respectively; p < 0.001). By the COX multivariate analysis, both overexpressed METTL3 and METTL14 were significantly associated with short overall survival (p < 0.05). Both METTL3 and METTL14 expressions and the m6 A amounts were significantly increased in HN6 (p < 0.05). Silencing of METTL3 and METTL14 in HN6 significantly inhibited cell proliferation (p < 0.01), but it failed to mitigate cell migration or invasion. CONCLUSIONS: METTL3 and METTL14 are overexpressed in OSCC tissues and in the HN6 OSCC cell line that promotes cell proliferation. Overexpressed METTL3 or METTL14 is found to be an independent prognostic factor for short overall survival in patients with OSCC.

Dental Anomalies in Ciliopathies: Lessons from Patients with BBS2, BBS7, and EVC2 Mutations.

Objective: To investigate dental anomalies and the molecular etiology of a patient with Ellis−van Creveld syndrome and two patients with Bardet−Biedl syndrome, two examples of ciliopathies. Patients and Methods: Clinical examination, radiographic evaluation, whole exome sequencing, and Sanger direct sequencing were performed. Results: Patient 1 had Ellis−van Creveld syndrome with delayed dental development or tooth agenesis, and multiple frenula, the feature found only in patients with mutations in ciliary genes. A novel homozygous mutation in EVC2 (c.703G>C; p.Ala235Pro) was identified. Patient 2 had Bardet−Biedl syndrome with a homozygous frameshift mutation (c.389_390delAC; p.Asn130ThrfsTer4) in BBS7. Patient 3 had Bardet−Biedl syndrome and carried a heterozygous mutation (c.389_390delAC; p.Asn130ThrfsTer4) in BBS7 and a homozygous mutation in BBS2 (c.209G>A; p.Ser70Asn). Her clinical findings included global developmental delay, disproportionate short stature, myopia, retinitis pigmentosa, obesity, pyometra with vaginal atresia, bilateral hydronephrosis with ureteropelvic junction obstruction, bilateral genu valgus, post-axial polydactyly feet, and small and thin fingernails and toenails, tooth agenesis, microdontia, taurodontism, and impaired dentin formation. Conclusions: EVC2, BBS2, and BBS7 mutations found in our patients were implicated in malformation syndromes with dental anomalies including tooth agenesis, microdontia, taurodontism, and impaired dentin formation.

Engineered osteoclasts resorb necrotic alveolar bone in anti-RANKL antibody-treated mice.

Medication-related osteonecrosis of the jaw (MRONJ) is a serious side effect of antiresorptive medications such as denosumab (humanized anti-RANKL antibody), yet its pathophysiology remains elusive. It has been posited that inhibition of osteoclastic bone resorption leads to the pathological sequelae of dead bone accumulation, impaired new bone formation, and poor wound healing in MRONJ, but this hypothesis has not been definitively tested. We previously engineered myeloid precursors with a conditional receptor activator of nuclear factor kappa-Β intracellular domain (iRANK cells), which differentiate into osteoclasts in response to a chemical inducer of dimerization (CID) independently of RANKL. In this study, we showed that CID-treated iRANK cells differentiated into osteoclasts and robustly resorbed mineralized surfaces even in the presence of anti-RANKL antibody in vitro. We then developed a tooth extraction-triggered MRONJ model in nude mice using anti-RANKL antibody to deplete osteoclasts. This model was used to determine whether reconstitution of engineered osteoclasts within sockets could prevent specific pathological features of MRONJ. Locally delivered iRANK cells successfully differentiated into multinucleated osteoclasts in response to CID treatment in vivo as measured by green fluorescent protein (GFP), tartrate-resistant acid phosphatase (TRAP), carbonic anhydrase II, matrix metallopeptidase 9 (MMP-9), and cathepsin K staining. Sockets treated with iRANK cells + CID had significantly more osteoclasts and less necrotic bone than those receiving iRANK cells alone. These data support the hypothesis that osteoclast deficiency leads to accumulation of necrotic bone in MRONJ.

Involvement of the A disintegrin and metalloproteinase 9 in oral cancer cell invasion.

The aims of this study were to determine the functional roles of the transmembrane glycoprotein, Disintegrin and metalloproteinase domain-containing protein 9 (ADAM 9), in the phosphorylation of epidermal growth factor receptor (EGFR) and AKT and in the aggressiveness of oral cancer cells. Immunohistochemistry and immunoblotting were conducted to determine expression of ADAM 9 and the levels of EGFR phosphorylated at the tyrosine 1173 residue (p-EGFRtyr1173 ) and AKT phosphorylated at the serine 473 residue (p-AKTser473 ) in oral cancer tissues and in the oral cancer cell lines HN5, HN6, HN15, and HN008. Small interfering RNA (siRNA) was used to inhibit expression of ADAM9 mRNA, and thus production of ADAM9 protein, in oral cancer cells. ADAM9-knockdown cells were examined for p-EGFRtyr1173 and p-AKTser473 levels and used for cell proliferation and invasion assays. A positive correlation among overexpression of ADAM 9, p-EGFRtyr1173 , and p-AKTser473 was found in oral cancer tissues. These biomolecules were also overexpressed in HN6 and HN15 cell lines. Expression of ADAM9 in HN6 and HN15 cells was statistically significantly inhibited by siRNA against ADAM9 mRNA (siADAM9) compared with the negative-control siRNA (scramble). The levels of p-AKTser473 , but not those of p-EGFRtyr1173 , were statistically significantly blocked by siADAM9. Although the proliferation rates of ADAM9 knocked-down HN6 and HN15 cells did not differ from those of cells exposed to scramble, a statistically significant decrease in cell invasion was found in these ADAM9-silenced cells. These results suggest a functional role of the ADAM 9/AKT signaling pathway in oral cancer cell invasion, which may be beneficial as a therapeutic target of oral cancer.

An in vitro culture system for long-term expansion of epithelial and mesenchymal salivary gland cells: role of TGF-ß1 in salivary gland epithelial and mesenchymal differentiation.

Despite a pivotal role in salivary gland development, homeostasis, and disease, the role of salivary gland mesenchyme is not well understood. In this study, we used the Col1a1-GFP mouse model to characterize the salivary gland mesenchyme in vitro and in vivo. The Col1a1-GFP transgene was exclusively expressed in the salivary gland mesenchyme. Ex vivo culture of mixed salivary gland cells in DMEM plus serum medium allowed long-term expansion of salivary gland epithelial and mesenchymal cells. The role of TGF-ß1 in salivary gland development and disease is complex. Therefore, we used this in vitro culture system to study the effects of TGF-ß1 on salivary gland cell differentiation. TGF-ß1 induced the expression of collagen, and inhibited the formation of acini-like structures in close proximity to mesenchymal cells, which adapted a fibroblastic phenotype. In contrast, TGF-ßR1 inhibition increased acini genes and fibroblast growth factors (Fgf-7 and Fgf-10), decreased collagen and induced formation of larger, mature acini-like structures. Thus, inhibition of TGF-ß signaling may be beneficial for salivary gland differentiation; however, due to differential effects of TGF-ß1 in salivary gland epithelial versus mesenchymal cells, selective inhibition is desirable. In conclusion, this mixed salivary gland cell culture system can be used to study epithelial-mesenchymal interactions and the effects of differentiating inducers and inhibitors.

Involvement of the P2X7 purinergic receptor and c-Jun N-terminal and extracellular signal-regulated kinases in cyclooxygenase-2 and prostaglandin E2 induction by LL-37.

Periodontal disease is caused by microorganisms and host-derived inflammation involving increased cyclooxygenase-2 (COX-2) expression and prostaglandin E(2) (PGE(2)) production. We previously demonstrated that human ß-defensin-3 induces COX-2 and PGE(2) in human gingival fibroblasts (HGFs). We, therefore, aimed to examine the inducible effects of LL-37, the only cathelicidin expressed in humans, on COX-2 expression and PGE(2) synthesis in HGFs and to elucidate the relevant signaling pathways. The COX-2 expression was upregulated by LL-37 in dose- and time-dependent manners. Accordingly, the synthesis of PGE(2) in cell-free culture supernatants was raised by LL-37 (p < 0.01) and blocked by NS-398, a specific COX-2 inhibitor (p < 0.01). P2X inhibitors and a neutralizing antibody against P2X(7) purinergic receptor significantly abrogated COX-2 induction and PGE(2) production by LL-37 (p < 0.01). LL-37 upregulated COX-2 expression and PGE(2) synthesis via activation of extracellular signal-regulated kinase (ERK) and p46 c-Jun N-terminal kinase (JNK), while interleukin-1ß did so via nuclear factor-ĸB and all three mitogen-activated protein kinases. In summary, LL-37 can control arachidonic acid metabolism by induction of COX-2 expression and PGE(2) synthesis via the P2X(7) receptor, ERK, and p46 JNK. The pro-inflammatory effects of LL-37 may be essential for initiating oral mucosal inflammation in periodontal disease.

An inducible, ligand-independent receptor activator of NF-κB gene to control osteoclast differentiation from monocytic precursors.

Osteoclasts are bone-resorbing cells that are critical for the normal formation and maintenance of teeth and skeleton. Osteoclast deficiency can contribute to heterotopic ossification (HO), a pathology that is particularly detrimental to the mechanical functions of joints, valves and blood vessels. On the other hand, osteoclast over-activity is a major cause of osteoporosis. A reliable method for controlled generation of osteoclasts would be useful as a potential autologous cell therapy for HO, as well as high-throughput drug screening for anti-osteoporotic drugs. In this report, we describe the development of a cell engineering approach to control monocytic precursor cell differentiation to osteoclasts. Oligomerization of receptor activator of nuclear factor κB (RANK) is known to be essential for osteoclast differentiation from monocyte/macrophage precursors. We engineered a murine monocytic cell line, RAW264.7 to express a fusion protein comprising the intracellular RANK signaling domain and FK506-derived dimerization domains that bind to a small molecule chemical inducer of dimerization (CID). Virally infected cells expressing this fusion protein were treated with CID and dose-dependent induction of tartrate-resistant acid phosphatase activity, as well as multinucleated osteoclast formation were observed. Furthermore, NF-κB signaling was upregulated in a CID-dependent fashion, demonstrating effective RANK intracellular signaling. Functionally CID-induced osteoclasts had robust mineral resorptive activity in both two-dimensional and three-dimensional in vitro resorption assays. In addition, the CID-induced osteoclasts have the same life span as native RANKL-induced osteoclasts. Most importantly and crucially, the engineered cells differentiated into osteoclasts that were resistant to the potent osteoclast inhibitor, osteoprotegerin. Taken together, these studies are the first to describe a method for inducible control of monocytic precursor differentiation to osteoclasts that may be useful for future development of an engineered autologous cell therapy as well as high-throughput drug testing systems to treat diseases of osteoclast over-activity that are independent of osteoprotegerin.