Consecutive intra-gingival injections of lipopolysaccharide and butyric acid to mice induce abnormal behavior and changes in cytokine concentrations.

BACKGROUND: Periodontopathic bacteria such as Porphyromonas gingivalis produce several metabolites, including lipopolysaccharide (LPS) and n-butyric acid (BA). Past work suggested that periodontal infection may cause cognitive impairment in mice. AIMS: To elucidate the mechanisms by which metabolites such as LPS and BA, resulting from Porphyromonas gingivalis activity, induce immunological and physiological abnormalities in mice. METHODS: In the present work, 28 male ICR mice were placed in an open-field arena and the total distance (cm/600 s) they covered was recorded. Based on their moving distances, mice were divided into 4 groups (n = 7) and injected the following substances into their gingival tissues for 32 consecutive days: saline (C), 5 mmol/L of BA (B), 1 µg/mouse of LPS (L), and BA-LPS (BL) solutions. Distances covered by mice were also measured on days 14 and 21, with their habituation scores considered as "(moving distance on day 14 or 21)/(moving distance on day 0)". Afterwards, mice were dissected, and hippocampal gene expression and the concentrations of short-chain fatty acids, neurotransmitters and cytokines in their blood plasma and brains were analyzed. In addition, mouse brain and liver tissues were fixed and visually assessed for histopathological abnormalities. RESULTS: Group BL had significantly higher habituation scores than C and B on day 14. LPS induced higher habituation scores on day 21. LPS induced significant decreases in the mRNA levels of interleukin (IL)-6 and brain-derived neurotrophic factors, and an increase in neurotrophic tyrosine kinase receptor type 2. In both plasma and brain, LPS induced a significant acetate increase. Moreover, LPS significantly increased acetylcholine in brain. In plasma alone, LPS and BA significantly decreased monocyte chemoattractant protein 1 (MCP-1). However, while LPS significantly decreased tyrosine, BA significantly increased it. Lastly, LPS significantly decreased IL-6 and tumor necrosis factor in plasma. No histopathological abnormalities were detected in liver or brain tissues of mice. CONCLUSION: We showed that injections of LPS and/or BA induced mice to move seemingly tireless and that both LPS and BA injections strongly induced a reduction of MCP-1 in blood plasma. We concluded that LPS and BA may have been crucial to induce and/or aggravate abnormal behavior in mice.

Varying butyric acid amounts induce different stress- and cell death-related signals in nerve growth factor-treated PC12 cells: implications in neuropathic pain absence during periodontal disease progression.

Neuropathic pain is absent from the early stages of periodontal disease possibly due to neurite retraction. Butyric acid (BA) is a periodontopathic metabolite that activates several stress-related signals and, likewise, induce neurite retraction. Neuronal cell death is associated to neurite retraction which would suggest that BA-induced neurite retraction is ascribable to neuronal cell death. However, the underlying mechanism of BA-related cell death signaling remains unknown. In this study, we exposed NGF-treated PC12 cells to varying BA concentrations [0 (control), 0.5, 1.0, 5.0 mM] and determined selected stress-related (H2O2, glutathione reductase, calcium (Ca(2+)), plasma membrane Ca(2+) ATPase (PMCA), and GADD153/CHOPS) and cell death-associated (extrinsic: FasL, TNF-α, TWEAK, and TRAIL; intrinsic: cytochrome C (CytC), NF-kB, CASP8, CASP9, CASP10, and CASP3) signals. Similarly, we confirmed cell death execution by chromatin condensation. Our results showed that low (0.5 mM) and high (1.0 and 5.0 mM) BA levels differ in stress and cell death signaling. Moreover, at periodontal disease-level BA concentration (5 mM), we observed that only FasL amounts were affected and occurred concurrently with chromatin condensation insinuating that cells have fully committed to neurodegeneration. Thus, we believe that both stress and cell death signaling in NGF-treated PC12 cells are affected differently depending on BA concentration. In a periodontal disease scenario, we hypothesize that during the early stages, low BA amounts accumulate resulting to both stress- and cell death-related signals that favor neurite non-proliferation, whereas, during the later stages, high BA amounts accumulate resulting to both stress- and cell death-related signals that favor neurodegeneration. More importantly, we propose that neuropathic pain absence at any stage of periodontal disease progression is ascribable to BA accumulation regardless of amount.

Varying hemin concentrations affect Porphyromonas gingivalis strains differently.

Porphyromonas gingivalis requires heme to grow, however, heme availability and concentration in the periodontal pockets vary. Fluctuations in heme concentration may affect each P. gingivalis strain differently, however, this was never fully demonstrated. Here, we elucidated the effects of varying hemin concentrations in representative P. gingivalis strains. Throughout this study, representative P. gingivalis strains [FDC381 (type I), MPWIb-01 (type Ib), TDC60 (type II), ATCC49417 (type III), W83 (type IV), and HNA99 (type V)] were used and grown for 24 h in growth media under varying hemin concentrations (5 × , 1 × , 0.5 × , 0.1 × ). Samples were lysed and protein standardized. Arg-gingipain (Rgp), H2O2, and superoxide dismutase (SOD) levels were subsequently measured. We focused our study on 24 h-grown strains which excluded MPWIb-01 and HNA99. Rgp activity among the 4 remaining strains varied with Rgp peaking at: 1 × for FDC381, 5 × for TDC60, 0.5 × for ATCC49417, 5 × and 0.5 × for W83. With regards to H2O2 and SOD amounts: FDC381 had similar H2O2 amounts in all hemin concentrations while SOD levels varied; TDC60 had the lowest H2O2 amount at 1 × while SOD levels became higher in relation to hemin concentration; ATCC49417 also had similar H2O2 amounts in all hemin concentrations while SOD levels were higher at 1 × and 0.5 × ; and W83 had statistically similar H2O2 and SOD amounts regardless of hemin concentration. Our results show that variations in hemin concentration affect each P. gingivalis strain differently.

Chaetocin inhibits RANKL-induced osteoclast differentiation through reduction of Blimp1 in Raw264.7 cells.

AIMS: Periodontitis is one of the most common bone-destructive diseases. Osteoclast is differentiated from hematopoietic macrophage-like cells through receptor activator of NFκB ligand (RANKL)-RANK signaling system, and the reduction in osteoclast formation may result in prevention of bone-resorptive diseases. Chaetocin is a compound isolated from fungal cultures and has been reported as a potent and selective inhibitor of suppressor of variegation 3-9 homolog 1 (Suv39h1), which catalyzes histone methylation on histone H3 lysine 9 (H3K9) residues. However, the effect of chaetocin on osteoclast differentiation is uncertain. In this study, we examine the effect of chaetocin on RANKL-induced osteoclast differentiation and cell growth. MAIN METHODS: Mouse macrophage-like Raw264.7 cells were treated with RANKL in the presence or absence of chaetocin, and tartrate-resistant acid phosphatase (TRAP) staining was performed. Cell growth was measured as the amount of DNA stained with SYTOX Green dye. Expression and production of osteoclast differentiation markers, anti-osteoclastogenic genes, B lymphocyte-induced maturation protein-1 (Blimp1), and cell growth suppressors were examined by qRT-PCR or/and Western blot analysis. KEY FINDINGS: Here we show that chaetocin dose-dependently reduced RANKL-induced osteoclast differentiation and cell growth via Blimp1 downregulation which results in the upregulation of osteoclast differentiation inhibitors and cell growth suppressors. These effects were not derived from the chaetocin's inhibitory effect of Suv39h1. SIGNIFICANCE: These results suggest that chaetocin suppresses RANKL-induced osteoclastogenesis and cell growth through blimp1 downregulation, followed by induction of anti-osteoclastogenic genes and cell growth suppressors, without inhibition of Suv39h1. Thus, chaetocin might be a drug candidate for the prevention of bone resorption in bone-destructive diseases.

Epstein-Barr virus infection in chronically inflamed periapical granulomas.

Periapical granulomas are lesions around the apex of a tooth caused by a polymicrobial infection. Treatment with antibacterial agents is normally performed to eliminate bacteria from root canals; however, loss of the supporting alveolar bone is typically observed, and tooth extraction is often selected if root canal treatment does not work well. Therefore, bacteria and other microorganisms could be involved in this disease. To understand the pathogenesis of periapical granulomas more precisely, we focused on the association with Epstein-Barr virus (EBV) using surgically removed periapical granulomas (n = 32). EBV DNA was detected in 25 of 32 periapical granulomas (78.1%) by real-time PCR, and the median number of EBV DNA copies was approximately 8,688.01/µg total DNA. In contrast, EBV DNA was not detected in healthy gingival tissues (n = 10); the difference was statistically significant according to the Mann-Whitney U test (p = 0.0001). Paraffin sections were also analyzed by in situ hybridization to detect EBV-encoded small RNA (EBER)-expressing cells. EBER was detected in the cytoplasm and nuclei of B cells and plasma cells in six of nine periapical granulomas, but not in healthy gingival tissues. In addition, immunohistochemical analysis for latent membrane protein 1 (LMP-1) of EBV using serial tissue sections showed that LMP-1-expressing cells were localized to the same areas as EBER-expressing cells. These data suggest that B cells and plasma cells in inflamed granulomas are a major source of EBV infection, and that EBV could play a pivotal role in controlling immune cell responses in periapical granulomas.

High butyric acid amounts induce oxidative stress, alter calcium homeostasis, and cause neurite retraction in nerve growth factor-treated PC12 cells.

Butyric acid (BA) is a common secondary metabolite by-product produced by oral pathogenic bacteria and is detected in high amounts in the gingival tissue of patients with periodontal disease. Previous works have demonstrated that BA can cause oxidative stress in various cell types; however, this was never explored using neuronal cells. Here, we exposed nerve growth factor (NGF)-treated PC1(2) cells to varying BA concentrations (0.5, 1.0, 5.0 mM). We measured total heme, H(2)O(2), catalase, and calcium levels through biochemical assays and visualized the neurite outgrowth after BA treatment. Similarly, we determined the effects of other common periodontal short-chain fatty acids (SCFAs) on neurite outgrowth for comparison. We found that high (1.0 and 5.0 mM) BA concentrations induced oxidative stress and altered calcium homeostasis, whereas low (0.5 mM) BA concentration had no significant effect. Moreover, compared to other SCFAs, we established that only BA was able to induce neurite retraction.

Ab initio modeling approach towards establishing the structure and docking orientation of the Porphyromonas gingivalis FimA.

Porphyromonas gingivalis FimA is a major aetiological agent in periodontal disease development, however, its structure has never been determined. Here, we established the mature P. gingivalis FimA ab initio model of all six FimA variants. We determined the conserved amino acid sequences of each FimA variant and generated mature FimA models. Subsequently, we validated their quality, protein empirical distribution, and radius of gyration. Similarly, structural comparison and topological orientation were elucidated, and the probable protein-protein docking was investigated. We found that the putative mature FimA model is ß-sheet-rich and, likewise, we observed that each mature FimA model has varying levels of structural differences which can be topologically subdivided into the upper, middle, and lower FimA sections. Moreover, we found that the FimA epithelial cell-binding domain (EBD) is structurally conserved within the middle FimA section of all variants and FimA-FimA docking suggests that the FimA EBDs are oriented in opposite and alternating directions of each other.

Similar physiological effects in Porphyromonas gingivalis ATCC 33277 under hemin-excess and hemin-limited concentrations are putatively associated to different hydrogen peroxide function.

Porphyromonas gingivalis requires optimal hemin to grow while non-optimal hemin hampers growth. Hemin induces H2O2 production while H2O2 has a dual function. In P. gingivalis ATCC 33277, we found similar physiological effects under hemin-excess and hemin-limited concentrations which we propose is related to two different functions of the H2O2 molecule.

High-sensitivity detection of short-chain fatty acids in porcine ileal, cecal, portal and abdominal blood by gas chromatography-mass spectrometry.

Short-chain fatty acids (SCFA), such as acetate, propionate and n-butyrate, are the main end-products of fermentation in the large intestine. SCFA are rapidly absorbed from the large intestinal mucosa to provide energy to the host. In this study, high-sensitivity detection of SCFA was demonstrated in blood using the gas chromatometry with mass spectrometry (GC-MS). Few studies have measured SCFA in porcine blood. Therefore, SCFA concentrations in the ileal (IV), cecal (CV), portal (PV) and abdominal (AV) vein blood, urine (Ur) and saliva (Sa) were measured by GC-MS. All body fluids were collected from four 5-month-old pigs. Cecal (CD) and ileal (ID) digesta, and cecal (CM) and ileal (IM) mucosa were also collected and their corresponding SCFA concentrations were measured using ion-exclusion high-performance liquid chromatography. GC-MS analyses were successful to determine the SCFA concentrations in the porcine body fluids. n-Butyrate concentration was surprisingly high in CV and its proportion remained higher in CV than that in CD and CM. Acetate showed a constantly high proportion in all porcine body fluids. Propionate was detected at a relatively high proportion in CV, IV and PV, but was low in AV.

Role of the histone H3 lysine 9 methyltransferase Suv39 h1 in maintaining Epsteinn-Barr virus latency in B95-8 cells.

The ability of Epstein-Barr Virus (EBV) to establish latent infection is associated with infectious mononucleosis and a number of malignancies. In EBV, the product of the BZLF1 gene (ZEBRA) acts as a master regulator of the transition from latency to the lytic replication cycle in latently infected cells. EBV latency is primarily maintained by hypoacetylation of histone proteins in the BZLF1 promoter by histone deacetylases. Although histone methylation is involved in the organization of chromatin domains and has a central epigenetic role in gene expression, its role in maintaining EBV latency is not well understood. Here we present evidence that the histone H3 lysine 9 (H3K9) methyltransferase suppressor of variegation 3-9 homolog 1 (Suv39 h1) transcriptionally represses BZLF1 in B95-8 cells by promoting repressive trimethylation at H3K9 (H3K9me3). Suv39 h1 significantly inhibited basal expression and ZEBRA-induced BZLF1 gene expression in B95-8 B cells. However, mutant Suv39 h1 lacks the SET domain responsible for catalytic activity of histone methyl transferase and thus had no such effect. BZLF1 transcription was augmented when Suv39 h1 expression was knocked down by siRNA in B95-8 cells, but not in Akata or Raji cells. In addition, treatment with a specific Suv39 h1 inhibitor, chaetocin, significantly enhanced BZLF1 transcription. Furthermore, chromatin immunoprecipitation assays revealed the presence of Suv39 h1 and H3K9me3 on nucleosome histones near the BZLF1 promoter. Taken together, these results suggest that Suv39 h1-H3K9me3 epigenetic repression is involved in BZLF1 transcriptional silencing, providing a molecular basis for understanding the mechanism by which EBV latency is maintained.

BIX01294 suppresses osteoclast differentiation on mouse macrophage-like Raw264.7 cells.

Gene expressionis controlled by epigenetic mechanisms including histone methylation. Osteoclasts are bone-resorptive cells that differentiate from hematopoietic-precursor cells by receptor activator of nuclear factor-κB ligand (RANKL) stimulation. Although BIX01294, a specific inhibitor of G9a, which works as a histone H3 lysine 9 (H3K9) methyltransferase, reportedly changes cellular differentiational stage, its effect on osteoclast differentiation is unclear. In this study, the effects of BIX01294 on osteoclast differentiation were examined. Here, we showed that BIX01294 dose-dependently reduced RANKL-induced tartrate-resistant acid phosphatase positive multinuclear osteoclast-like cell differentiation from murine macrophage-like Raw264.7 cells. During differentiation, growth rates reduced only less than 14% of those of cells stimulated with RANKL alone by BIX01294 treatment. Moreover, western blot analysis showed that BIX01294 reduced RANKL-induced carbonic anhydrase II and cathepsin K production and decreased RANKL-induced nuclear factor of activated T-cell c1, a master regulatory transcription factor, production during osteoclast differentiation. These results suggest that BIX01294 suppresses RANKL-induced osteoclast differentiation. This is the first report about the effect of BIX01294 on osteoclast differentiation.

Higher prevalence of Epstein-Barr virus DNA in deeper periodontal pockets of chronic periodontitis in Japanese patients.

Periodontitis, a complex chronic inflammatory disease caused by subgingival infection, is among the most prevalent microbial diseases in humans. Although traditional microbiological research on periodontitis has focused on putative bacteria such as Porphyromonas gingivalis, the herpes virus is proposed to be involved in the pathogenesis of periodontitis because bacterial etiology alone does not adequately explain various clinical aspects. In this study, we established for the first time, more Epstein-Barr virus (EBV) DNA is found deeper in periodontal pockets of chronic periodontitis in Japanese patients. Subgingival samples were collected from 85 patients with chronic periodontitis having two periodontal sites with probing depths (PD) of ≤ 3 mm (shallow) or ≥ 5 mm (deep) and were subjected to a nested polymerase chain reaction. EBV DNA was more frequently detected in patients with deeper PD sites (66%) than in those with shallow PD sites (48%) or healthy controls (45%). Coexistence of EBV DNA and P. gingivalis was significantly higher in patients with deeper PD sites (40%) than in those with shallow PD sites (14%) or healthy controls (13%). Although no difference in clinical index for periodontitis, the odds ratio of EBV DNA in patients with deeper PD sites was 2.36, which was 2.07-fold higher than that in those with shallow PD sites. Interestingly, the odds of acquiring chronic periodontitis (PD ≥ 5 mm) were higher in the presence of both EBV DNA and P. gingivalis compared with either EBV DNA or P. gingivalis only. In addition, we also observed that EBV-encoded small RNA (EBER) in positive cells of human gingival tissues. These results would suggest that EBV DNA may serve as a pathogenic factor leading to chronic periodontitis among Japanese patients.

Catechin inhibits Candida albicans dimorphism by disrupting Cek1 phosphorylation and cAMP synthesis.

Candida albicans is a fungal pathogen that undergoes dimorphism (transformation from a yeast form to a hyphal form), wherein, the yeast form is identified as a disseminating form that plays a critical role in the early stages of Candida disease progression, while the hyphal form is found to exert additional pathogenicity by adapting to various environmental conditions. Here, we elucidated the effects of catechin on C. albicans hyphal formation. Flow cytometry analysis showed catechin inhibited FCS-induced hyphal formation. Moreover, hypha-specific gene expression in MAP kinase cascade and cAMP pathway was decreased ascribable to catechin. Furthermore, through Western blotting and cAMP synthesis analysis, we found catechin obstructs Cek1 phosphorylation in MAP kinase cascade and suppresses cAMP synthesis. These results suggest that catechin possesses anti-dimorphism activity by interfering with in vitro signal transduction. Similarly, this highlights the possible application of catechin in clinical therapy for the management and prevention of candidosis.

Vaspin attenuates RANKL-induced osteoclast formation in RAW264.7 cells.

Visceral adipose tissue-derived serine protease inhibitor (vaspin), an adipokine that was recently identified in a rat model of type 2 diabetes, has been suggested to have an insulin-sensitizing effect. In this study, we investigated whether vaspin inhibits receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclastogenesis using two types of osteoclast precursors: RAW264.7 cells and bone marrow cells (BMCs). Vaspin inhibited RANKL-induced osteoclastogenesis in RAW264.7 cells and BMCs. Interestingly, vaspin also inhibited the RANKL-induced expression of nuclear factor of activated T cells c1 (NFATc1) in RAW264.7 cells and BMCs. Furthermore, it inhibited the RANKL-induced upregulation of matrix metalloproteinase-9 and cathepsin K in RAW264.7 cells. Thus, we suggest that vaspin downregulates osteoclastogenesis in part by inhibiting expression of the transcription factor NFATc1.

Activation-induced cytidine deaminase mRNA expression in oral squamous cell carcinoma-derived cell lines is upregulated by inflammatory cytokines.

Activation-induced cytidine deaminase (AID) induces cytosine deamination to generate somatic hypermutation and class switch recombnation in immunoglobulin genes. AID expression is upregulated by inflammatory cytokines such as interferon-γ and tumor necrosis factor (TNF)-α, which in turn induce p53 mutations in inflammatory or cancer cells. In this study, the effects of growth factors, cytokines or sodium butyrate on AID mRNA expression were examined in human OSCC-derived cells using real-time RT-PCR. Expression of AID mRNA was detected in OSCC cells and the expression was increased by EGF, TNF-a, or sodium butyrate. These results suggest that aberrant AID expression may play an important role in the dysplasia-carcinoma sequence in the oral cavity.

The periodontal pathogen Porphyromonas gingivalis induces the Epstein-Barr virus lytic switch transactivator ZEBRA by histone modification.

Epstein-Barr virus (EBV) is a ubiquitous human herpesvirus that usually results in latent infection of B cells. The EBV BZLF1 gene product ZEBRA is a master regulator of the transition from latency to the lytic replication cycle. In the latent state, hypoacetylation of histone proteins in the BZLF1 promoter by histone deacetylases (HDACs) is primarily involved in maintaining EBV latency. Although the mechanism that regulates the switch between latency and lytic replication has been a central research focus in EBV infection, the causal link between HDAC inhibition and the disruption of viral latency is not well understood. Periodontal disease is a complex chronic inflammatory disease caused by subgingival infection with oral anaerobic bacteria, typically Porphyromonas gingivalis. Periodontal disease occurs worldwide and is among the most prevalent microbial diseases in humans. In this study, we examined the biological effect of P. gingivalis infection on EBV reactivation and found that P. gingivalis induced expression of ZEBRA. This activity was associated with supernatant from bacterial culture, but not with other bacterial components such as lipopolysaccharide or fimbriae. We demonstrated that culture supernatant from P. gingivalis, which contained high concentrations of butyric acid, inhibited HDACs, thus increasing histone acetylation and the transcriptional activity of the BZLF1 gene. Chromatin immunoprecipitation assays revealed that HDACs were present in the BZLF1 promoter during latent state and that they were dissociated from the promoter concomitantly with the association of acetylated histone H3, upon stimulation by culture supernatant from P. gingivalis. Thus, P. gingivalis induced EBV reactivation via chromatin modification, and butyric acid-a bacterial metabolite-was responsible for this effect. These findings suggest that periodontal disease is a risk factor for EBV reactivation in infected individuals and might therefore contribute to progression of EBV-related diseases.

Role of histone modification on transcriptional regulation and HIV-1 gene expression: possible mechanisms of periodontal diseases in AIDS progression.

Although approximately 200 distinct cell types - including fibroblasts, neurons, and hematopoietic cells - possess the same DNA sequence, they have diverse functions in humans and exhibit considerably different gene expression patterns. It has become increasingly clear that epigenetic regulation plays an important role in gene expression. There are two major forms of epigenetic regulation: posttranslational modification of DNA-associated histone proteins in chromatin and methylation of DNA. These forms are regulated by distinct but coupled pathways. Notably, histone Lys acetylation by histone acetyltransferase and deacetylation by histone deacetylases play a crucial role in on-off regulation of gene expression. It is now understood that epigenetics plays an important role not only in the regulation of gene expression but also in the pathogenesis of a broad range of diseases such as cancer and microbial infections. We have determined that epigenetic regulation is involved in the establishment and maintenance of HIV-1 latency and in the reactivation of HIV-1 by periodontopathic bacteria. In this review, we focus on the effect of histone modification on transcriptional regulation and the contribution thereof to the regulation of HIV-1 gene expression during the lytic and latent stages of HIV-1 infection. Likewise, we discuss the mechanisms by which periodontal diseases may accelerate AIDS progression in infected individuals as a new systemic disease caused by periodontitis and describe potential therapeutic interventions based on epigenetic mechanisms.

Inhibitory effects of a tryptamine derivative on ultraviolet radiation-induced apoptosis in MC3T3-E1 mouse osteoblasts.

MS-IPA1 is a new synthetic compound that is synthesized from tryptamine. Recently, our group demonstrated that SST-VED-I-1, which has a similar chemical structure to MS-IPA1, inhibits starvation-induced apoptosis in osteoblasts. However, the effects of MS-IPA1 on apoptosis in osteoblasts have not yet been examined. Therefore, this study examined the effects of this compound on apoptosis in osteoblasts. In this study, MC3T3-E1 mouse osteoblasts were used and apoptosis was induced by ultraviolet radiation (UV). We investigated the effect of MS-IPA1 on apoptosis by analyzing caspase3/7 activity, translocation of phosphatidylserine (PS), and mRNA expression levels of Bcl-2 and Bax. In addition, it was investigated whether MS-IPA1 affects cell proliferation and cell cycle progression. We found that MS-IPA1 had no effect on cell proliferation or cell cycle progression. However, MS-IPA1 suppressed UV-induced cell death in a dose-dependent manner, which was accompanied with the inhibition of caspase activation and translocation of PS. Furthermore, after UV exposure, Bcl-2 expression was increased in the MS-IPA1-treated cells as compared to that in the vehicle-treated cells. In contrast, Bax expression was decreased in the MS-IPA1-treated cell as compared to that in the vehicle-treated cells. These results suggest that MS-IPA1 has an inhibitory effect on apoptosis in osteoblasts through a Bcl-2 family-dependent signaling pathway.

Interleukin-17A induces cathepsin K and MMP-9 expression in osteoclasts via celecoxib-blocked prostaglandin E2 in osteoblasts.

Interleukin-17 (IL-17) is a cytokine secreted primarily by T(H)-17 cells that can stimulate the development of osteoclasts (osteoclastogenesis) in the presence of osteoblasts. IL-17, through osteoblasts, has indirect effects on the expression of bone resorption-related enzymes in osteoclasts, which have not been well clarified. Here, using MC3T3-E1 cells and RAW264.7 cells as osteoblasts and osteoclast precursors, we aimed to clarify these effects of IL-17A. MC3T3-E1 cells were cultured in the presence or absence of IL-17A for 72 h and the conditioned media collected (in the presence of soluble receptor activator of NF-кB ligand) and used to culture RAW264.7 cells. To assess osteoclast differentiation, adherent cells were fixed and stained for tartrate-resistant acid phosphatase (TRAP). Our analyses demonstrated that the number of TRAP-positive multinucleated cells increases after 3 days of culture in conditioned medium from IL-17A-treated cells compared to untreated controls. In addition, we observed that the levels of cathepsin K and MMP-9 increase in the conditioned medium from IL-17A-treated cells, whereas CA II expression levels remain unaffected. PGE(2) production from MC3T3-E1 cells increased in the presence of IL-17A. Celecoxib, a specific inhibitor of cyclooxygenase-2 (COX-2), blocked both the IL-17A-stimulated increase in TRAP-positive multinucleated cells and the expression of cathepsin K and MMP-9. Furthermore, when MC3T3-E1 cells were transformed with small interfering RNA to silence COX-2 expression before IL-17A treatment, the resulting conditioned medium was less effective at inducing cathepsin K and MMP-9 expression in RAW264.7 cells. These results suggest that IL-17A induces the differentiation and function of osteoclasts via celecoxib-blocked prostaglandin, mainly PGE(2), in osteoblasts.

Inhibitory Effects of a Tryptamine Derivative on Ultraviolet Radiation-Induced Apoptosis in MC3T3-E1 Mouse Osteoblasts.

MS-IPA1 is a new synthetic compound that is synthesized from tryptamine. Recently, our group demonstrated that SST-VED-I-1, which has a similar chemical structure to MS-IPA1, inhibits starvation-induced apoptosis in osteoblasts. However, the effects of MS-IPA1 on apoptosis in osteoblasts have not yet been examined. Therefore, this study examined the effects of this compound on apoptosis in osteoblasts. In this study, MC3T3-E1 mouse osteoblasts were used and apoptosis was induced by ultraviolet radiation (UV). We investigated the effect of MS-IPA1 on apoptosis by analyzing caspase3/7 activity, translocation of phosphatidylserine (PS), and mRNA expression levels of Bcl-2 and Bax. In addition, it was investigated whether MS-IPA1 affects cell proliferation and cell cycle progression. We found that MS-IPA1 had no effect on cell proliferation or cell cycle progression. However, MS-IPA1 suppressed UV-induced cell death in a dose-dependent manner, which was accompanied with the inhibition of caspase activation and translocation of PS. Furthermore, after UV exposure, Bcl-2 expression was increased in the MS-IPA1-treated cells as compared to that in the vehicle-treated cells. In contrast, Bax expression was decreased in the MS-IPA1-treated cell as compared to that in the vehicle-treated cells. These results suggest that MS-IPA1 has an inhibitory effect on apoptosis in osteoblasts through a Bcl-2 family-dependent signaling pathway.