Suttichai Krisanaprakornkit

Involvement of P2X7 purinergic receptor and MEK1/2 in interleukin-8 up-regulation by LL-37 in human gingival fibroblasts

BACKGROUND AND OBJECTIVE The antimicrobial peptide LL-37, derived from human neutrophils, can directly chemoattract leukocytes and up-regulate the expression of several immune-related genes in various cell types. In this study, we wanted to determine the immunoregulatory effect of LL-37 on interleukin-8 (IL-8) expression in human gingival fibroblasts (HGFs) and to characterize intracellular signaling pathway(s) and receptor(s) involved in IL-8 induction. MATERIAL AND METHODS Cultured fibroblasts were treated with different concentrations of LL-37 or interleukin-1β (IL-1β), as a positive control, for specific periods of time in the presence or absence of various inhibitors. RT-PCR and real-time PCR were conducted to analyze the expression of IL-8 mRNA, and the IL-8 levels in cell-free culture media were measured using ELISAs. The MTT assay was performed to determine the cytotoxicity of LL-37. RESULTS Nontoxic concentrations of LL-37 (up to 10 μm) and IL-1β significantly up-regulated the expression of IL-8 mRNA in a dose-dependent manner (p < 0.05). The IL-8 protein levels were consistently significantly elevated in conditioned media of LL-37-treated HGFs (p < 0.05). IL-8 up-regulation by LL-37 was completely abrogated by 20 μm U0126, consistent with transient phosphorylation of p44/42 MAP kinases. Moreover, pretreatment with Brilliant Blue G (a selective antagonist of the P2X(7) receptor) and the neutralizing antibody against P2X(7) blocked IL-8 up-regulation in a dose-dependent manner, consistent with expression of the P2X(7) receptor in HGFs. CONCLUSION These findings indicate that LL-37 induces IL-8 expression via the P2X(7) receptor and the MEK1/2-dependent p44/42 MAP kinases in HGFs, suggesting both direct and indirect involvement of LL-37 in neutrophil recruitment into an inflammatory site within diseased periodontal tissues.

Epithelial-Mesenchymal Transition in Oral Squamous Cell Carcinoma

Oral cancer is one of the drastic human cancers due to its aggressiveness and high mortality rate. Of all oral cancers, squamous cell carcinoma is the most common accounting for more than 90%. Epithelial-mesenchymal transition (EMT) is suggested to play an important role during cancer invasion and metastasis. Recently, emerging knowledge on EMT in carcinogenesis is explosive, tempting us to analyze previous studies on EMT in oral squamous cell carcinoma (OSCC). In this paper, we have first addressed the general molecular mechanisms of EMT, evidenced by alterations of cell morphology during EMT, the presence of cadherin switching, turning on and turning off of many specific genes, the activation of various signaling pathways, and so on. The remaining part of this paper will focus on recent findings of the investigations of EMT on OSCC. These include the evidence of EMT taking place in OSCC and the signaling pathways employed by OSCC cells during their invasion and metastasis. Collectively, with the large body of new knowledge on EMT in OSCC elaborated here, we are hopeful that targeting treatment for OSCC will be developed.

Overexpression and activation of Akt2 protein in oral squamous cell carcinoma

Aberrations of signal transducers in PI3K/Akt pathway have been found in many human cancers, and may play a critical role in carcinogenesis. Advanced research on oral cancer treatments, using novel agents targeting the PI3K/Akt signaling pathway, is being investigated with promising results. The objectives of this study were (1) to investigate expression of pan-Akt and its phosphorylated form (p-Akt), Akt1, and Akt2 in oral squamous cell carcinoma (OSCC) specimens (n=20) by immunohistochemistry, and (2) to determine mRNA expression of three Akt isoforms, including Akt1, Akt2, and Akt3, as well as their respective proteins, in five oral cancer cell lines and normal human oral keratinocytes (HOKs) by RT-PCR and Western blot assays. The results show that pan-Akt was expressed in 80% of OSCC cases, while Akt1, Akt2, and p-Akt were expressed in all OSCC cases. An intense expression of p-Akt at the invasive fronts of some OSCC samples was observed. Consistent with the immunohistochemical findings, p-Akt and Akt2 were overexpressed in all oral cancer cell lines in comparison with HOKs, whereas Akt2 mRNA was constitutively expressed, suggesting post-transcriptional regulation. In contrast, Akt1 mRNA and protein were constitutively expressed in all oral cancer cell lines and HOKs, while Akt3 mRNA appeared to be minimally expressed. In summary, these findings demonstrate that Akt2 and p-Akt are overexpressed in OSCC and may be involved in carcinogenesis, and suggest that post-transcriptional modification of Akt2 in OSCC may occur.

The Antimicrobial Peptide, LL-37, Inhibits in vitro Osteoclastogenesis

Uncoupled bone resorption leads to net alveolar bone loss in periodontitis. The deficiency of LL-37, the only human antimicrobial peptide in the cathelicidin family, in patients with aggressive periodontitis suggests that LL-37 may play a pivotal role in the inhibition of alveolar bone destruction in periodontitis. We aimed to investigate a novel function of LL-37 in osteoimmunity by blocking osteoclastogenesis in vitro. Human osteoclast progenitor cells were isolated from a buffy coat of blood samples. The cells were cultured in the presence of various concentrations of LL-37 during an in vitro induction of osteoclastogenesis. Non-toxic doses of LL-37 could block multinuclear formation of the progenitor cells and significantly diminish the number of tartrate-resistant acid-phosphatase-positive cells and the formation of resorption pits (p < 0.05), whereas these concentrations induced cellular proliferation, as demonstrated by increased expression of proliferating cell nuclear antigen. Expression of several osteoclast genes was down-regulated by LL-37 treatment. It was demonstrated that nuclear translocation of nuclear-factor-activated T-cells 2 (NFAT2) was blocked by LL-37 treatment, consistent with a significant reduction in the calcineurin activity (p < 0.005). Collectively, our findings demonstrate that LL-37 inhibits the in vitro osteoclastogenesis by inhibiting the calcineurin activity, thus preventing nuclear translocation of NFAT2. Abbreviations: CALCR, calcitonin receptor; ClC-7, chloride-proton exchanger; CTSK, cathepsin K; DAPI, 4′,6-diamidino-2-phenylindole; EGTA, ethylene glycol tetraacetic acid; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; M-CSF/CSF1, macrophage-colony- stimulating factor; MMP-9, matrix metalloproteinase-9; MTT, [3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]; NFAT2, nuclear factor of activated T-cells 2; PBS, phosphate-buffered saline; PCNA, proliferating cell nuclear antigen; PCR, polymerase chain reaction; RANK, receptor activator of nuclear factor kappa-B; RANKL, receptor activator of nuclear factor kappa-B ligand; RT-PCR, reverse-transcription polymerase chain-reaction; TBS, Tris-buffered saline; TCIRG1, T-cell, immune regulator 1, ATPase, H+ transporting, lysosomal V0 subunit A3; TRAcP, tartrate-resistant acid phosphatase.

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 E2 (PGE2) production. We previously demonstrated that human β-defensin-3 induces COX-2 and PGE2 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 PGE2 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 PGE2 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 P2X7 purinergic receptor significantly abrogated COX-2 induction and PGE2 production by LL-37 (p < 0.01). LL-37 upregulated COX-2 expression and PGE2 synthesis via activation of extracellular signal-regulated kinase (ERK) and p46 c-Jun N-terminal kinase (JNK), while interleukin-1β 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 PGE2 synthesis via the P2X7 receptor, ERK, and p46 JNK. The pro-inflammatory effects of LL-37 may be essential for initiating oral mucosal inflammation in periodontal disease.

Human β‐defensin‐3 up‐regulates cyclooxygenase‐2 expression and prostaglandin E2 synthesis in human gingival fibroblasts

BACKGROUND AND OBJECTIVE Oral epithelial cells express three antimicrobial peptide human beta-defensins (hBDs) that have previously been demonstrated to exert proinflammatory effects on various immune cells. We wanted to examine whether hBDs could induce cyclooxygenase-2 (COX-2) expression and prostaglandin E(2) (PGE(2)) synthesis in non-immune cells, such as human gingival fibroblasts. MATERIAL AND METHODS Cultured fibroblasts were treated with different concentrations of hBD-1, -2, -3 or interleukin-1 beta, as a positive control, for various times, in the presence or absence of NS-398, a specific COX-2 inhibitor. The levels of COX-1 and COX-2 mRNA expression were analyzed using RT-PCR and real-time PCR. Whole cell lysates were analyzed for COX-1 and COX-2 protein expression by western blotting. Cell-free culture supernatants were assayed for PGE(2) levels by ELISA. The lactate dehydrogenase assay was performed to determine the cytotoxicity of hBDs. RESULTS Ten and 40 microg/mL of hBD-3 up-regulated COX-2 mRNA and protein expression, consistent with COX-2 up-regulation by interleukin-1 beta, whereas hBD-1 and hBD-2 did not. However, COX-1 mRNA and protein were constitutively expressed. The time-course study revealed that hBD-3 up-regulated COX-2 mRNA and protein expression at 6 and 12 h, respectively. Consistent with COX-2 up-regulation, 10 and 40 microg/mL of hBD-3 significantly increased PGE(2) levels in cell-free culture supernatants (p < 0.05), and this was inhibited by NS-398 in a dose-dependent manner. Neither of the hBD concentrations tested in this study was toxic to the cells. CONCLUSION These findings indicate that epithelial human beta-defensin-3 functions as a proinflammatory mediator in controlling arachidonic acid metabolism in underlying fibroblasts.

Raised chondroitin sulphate WF6 epitope levels in gingival crevicular fluid in chronic periodontitis.

AIM To determine the levels of chondroitin sulphate (CS) WF6 epitope, recognized by WF6 monoclonal antibody, in gingival crevicular fluid (GCF) from different stages of periodontal disease and healthy periodontium, and to correlate those levels with clinical parameters. MATERIAL AND METHODS GCF samples, collected from 389 sites, were analysed for the WF6 epitope levels by the competitive enzyme-linked immunosorbent assay. RESULTS The median WF6 epitope level was significantly higher in chronic periodontitis sites (n=185) than in healthy and gingivitis sites (n=204) (p<0.001), whereas the median levels did not significantly differ between healthy (n=65) and gingivitis sites (n=139). The median level in severe periodontitis sites (n=60) was significantly higher than that in moderate periodontitis sites (n=63) (p=0.019). Similarly, the median level in moderate periodontitis sites was significantly higher than that in slight periodontitis sites (n=62) (p=0.001). The WF6 epitope levels significantly correlated with probing depth (r=0.777, p=0.001) and loss of clinical attachment level (r=0.814, p=0.001). CONCLUSION Elevated CS WF6 epitope levels in GCF are associated with severity of periodontitis. The WF6 antibody may therefore be clinically applied to monitor disease severity and progression.

Positive correlations between hCAP18/LL‐37 and chondroitin sulphate levels in chronic periodontitis

AIM To measure the levels of hCAP18/LL-37 in gingival crevicular fluid from patients with periodontal diseases compared with healthy controls and to determine the correlation between hCAP18/LL-37 and chondroitin sulphate (CS) levels in patients with periodontitis. MATERIAL AND METHODS Gingival crevicular fluid samples from 51 patients and 25 healthy volunteers were analysed for the hCAP18/LL-37 levels by immunoblotting and were determined for the CS levels by the competitive enzyme-linked immunosorbent assay. RESULTS Tris buffer pH 9.85 was selected to recover hCAP18/LL-37 from Periopaper strips, in which the percentages of recovery were around 70%. The median levels of hCAP18/LL-37 in the aggressive and the chronic periodontitis (CP) groups were significantly greater than those in the gingivitis and the healthy groups (p < 0.05). Significant correlations between the unprocessed 18-kDa fragment and CS levels (r = 0.650; p < 0.001) and between the mature 4.6-kDa fragment and CS levels (r = 0.502; p < 0.001) were observed only in the CP group. CONCLUSION The significant correlations between the hCAP18/LL-37 and the CS levels were found in CP, but not in aggressive periodontitis. The presence versus absence of such correlations may be clinically applicable to help clinicians distinguish between two distinct types of periodontitis.

Comparisons of the chondroitin sulphate levels in orthodontically moved canines and the clinical outcomes between two different force magnitudes.

The aims of this study were to compare the chondroitin sulphate (CS) levels in gingival crevicular fluid (GCF) of moved canines using either 70 or 120 g of orthodontic force, and to compare the rate of tooth movement and the amount of pain between these two force magnitudes. Sixteen patients (6 males and 10 females; aged 16.91 ± 2.99 years), with class I malocclusion, who required orthodontic treatment with first premolar extractions, were recruited. The force magnitudes used to move the maxillary canines distally were controlled at 70 and 120 g on the right and the left sides, respectively. GCF samples were collected with Periopaper(®) strips before and during orthodontic tooth movement. Competitive ELISA with monoclonal antibody was used to measure the CS levels. The distance of tooth movement and the amount of pain assessed by visual analog scale (VAS) scores were evaluated. The medians of CS levels during the loaded period were significantly greater than those during the unloaded period (P < 0.05). The differences between the medians of CS levels of 70 g and 120 g retraction force during each 1 week period were not significant. There was no significant difference in the rates of canine movement between these two force magnitudes. However, using 120 g, the medians of VAS scores were significantly greater than those with 70 g (P < 0.05). Collectively, 70 g retraction force appears to be sufficient and more suitable than 120 g force as it causes no difference in biochemically-assessed bone remodelling activity, the same rate of tooth movement, reduced pain and better comfort.

CD99 ligation induces intercellular cell adhesion molecule-1 expression and secretion in human gingival fibroblasts

OBJECTIVE To examine CD99 expression and its functional role in ICAM-1 induction in human gingival fibroblasts (HGFs) and human gingival epithelial cells (HGECs) by activating cells with anti-CD99 monoclonal antibody, MT99/3. BACKGROUND Engagement of CD99 with agonistic antibodies has been shown to regulate immune responses, cell adhesion and migration, and cell death in several studies. Particularly, this engagement results in transendothelial migration of leukocytes mediated by intercellular adhesion molecule-1 (ICAM-1) induction in endothelial cells. METHODS Total mRNA and protein were isolated from HGFs and HGECs for analyses of CD99 and ICAM-1 expression. Surface expression of CD99 and ICAM-1 was analysed by flow cytometry, and the detection of soluble ICAM-1 was assayed by immunoprecipitation and ELISA. RESULTS CD99 surface expression was constitutive on HGFs to a greater extent than that on HGECs. CD99 ligation with MT99/3 induced ICAM-1 mRNA expression in HGFs, but not in HGECs. Interestingly, CD99 ligation led to an increased level of soluble ICAM-1 detected in culture supernatant, whereas interleukin-1β (IL-1β) treatment induced expression of membrane-bound ICAM-1. Furthermore, ICAM-1 induction by CD99 engagement was demonstrated to involve the activation of the p50 subunit of nuclear factor-kappaB (NF-κB), extracellular signal-regulated kinase, and p46 c-Jun N-terminal kinase that differed from that by IL-1β treatment. CONCLUSION Our study has shown the involvement of CD99 ligation in the up-regulation of ICAM-1 expression and its secretion in gingival fibroblasts, which may be essential for better understanding of the pathogenesis of periodontal disease.