Ok-Jin Park

FGF2-activated ERK Mitogen-activated Protein Kinase Enhances Runx2 Acetylation and Stabilization

Runx2 is a key transcription factor regulating osteoblast differentiation and skeletal morphogenesis, and FGF2 is one of the most important regulators of skeletal development. The importance of the ERK mitogen-activated protein (MAP) kinase pathway in cranial suture development was demonstrated by the findings that the inhibition of FGF/FGF receptor (FGFR) signaling by a MEK blocker prevents the premature suture closure caused by an Fgfr2 mutation in mice. We previously demonstrated that ERK activation does not affect Runx2 gene expression but that it stimulates Runx2 transcriptional activity. However, the molecular mechanism underlying Runx2 activation by FGF/FGFR or ERK was still unclear. In this study, we found that FGF2 treatment increased the protein level of exogenously overexpressed Runx2 and that this increase is reversed by ERK inhibitors. In contrast, overexpression of constitutively active MEK strongly increased the Runx2 protein level, which paralleled an increase in Runx2 acetylation. As Runx2 protein phosphorylation mediated by ERK directly correlates with Runx2 protein stabilization, acetylation, and ubiquitination, we undertook to identify the ERK-dependent phosphorylation sites in Runx2. Analysis of two C-terminal Runx2 deletion constructs showed that the middle third of the protein is responsible for ERK-induced stabilization and activation. An in silico analysis of highly conserved ERK targets indicated that there are three relevant serine residues in this domain. Site-directed mutagenesis implicated Ser-301 in for ERK-mediated Runx2 stabilization and acetylation. In conclusion, the FGF2-induced ERK MAP kinase strongly increased the Runx2 protein level through an increase in acetylation and a decrease in ubiquitination, and these processes require the phosphorylation of Runx2 Ser-301 residue.

Interaction of Fas Ligand and Fas Expressed on Osteoclast Precursors Increases Osteoclastogenesis

We incidentally found that osteoclast precursors and mature osteoclasts express Fas ligand (FasL) as well as Fas, which was confirmed by flow cytometry, immunofluorescent staining, and RT-PCR. The aim of this study was to determine the role of FasL in differentiation and cell death of osteoclasts. To study the role of FasL in osteoclastogenesis, neutralizing anti-FasL mAb or rFasL was added during receptor activator of NF-κB ligand (RANKL)-induced osteoclastogenesis using bone marrow-derived macrophages. Neutralization of endogenous FasL by anti-FasL mAb decreased osteoclastogenesis, whereas rFasL enhanced osteoclast differentiation in a dose-dependent manner. In addition, rFasL up-regulated the secretion of osteoclastogenic cytokines, such as IL-1β and TNF-α, and the activation of NF-κB. Functional blocking of IL-1β and TNF-α using IL-1 receptor antagonist and soluble TNFR confirmed that those cytokines mediated the effect of FasL on osteoclastogenesis. The osteoclast precursors were relatively resistant to rFasL-induced apoptosis especially before RANKL treatment, resulting in minimal cell loss by rFasL treatment during osteoclastogenesis. Although rFasL increased the cell death of mature osteoclasts, growth factor withdrawal induced much more cell death. However, anti-FasL mAb did not affect the survival of mature osteoclasts, suggesting that the endogenous FasL does not have a role in the apoptosis of osteoclasts. Finally, in contrast to the effect on apoptosis, rFasL-assisted osteoclastogenesis was not mediated by caspases. In conclusion, FasL has a novel function in bone homeostasis by enhancing the differentiation of osteoclasts, which was not considered previously.

Pyrosequencing Analysis of Subgingival Microbiota in Distinct Periodontal Conditions

Subgingival microorganisms are potentially associated with periodontal diseases. However, changes in the subgingival microbiota during the progress of periodontal diseases are poorly understood. In this study, we analyzed bacterial communities in the subgingival paper point samples from 32 Korean individuals with no sign of disease, gingivitis, or periodontitis using 454 FLX Titanium pyrosequencing. A total of 256,113 reads representing 26 phyla, 433 genera, and 1,016 species were detected. Bacteroidetes, Fusobacteria, Synergistetes, and Spirochaetes were the abundant phyla in periodontitis subjects, whereas Firmicutes and Proteobacteria were identified as the dominant phyla in the gingivitis and healthy subjects, respectively. Although high levels of Porphyromonas, Fusobacterium, Fretibacterium, Rothia, Filifactor, and Treponema genera were observed in the periodontitis subjects, Streptococcus, Capnocytophaga, Leptotrichia, and Haemophilus genera were found at high frequency in the gingivitis subjects. Species including Porphyromonas gingivalis, Fusobacterium nucleatum, and Fretibacterium fastidiosum were significantly increased in periodontitis subjects. On the other hand, Streptococcus pseudopneumoniae, Haemophilus parainfluenzae, and Leptotrichia hongkongensis were preferentially observed in the gingivitis subjects. Intriguingly, the halophile Halomonas hamiltonii was revealed as a predominant species in the healthy subjects. Based on Fast UniFrac analysis, distinctive bacterial clusters were classified for the healthy, gingivitis, and periodontitis state. The current findings might be useful for understanding the pathogenesis, diagnosis, and treatment of periodontal diseases.

Lipoproteins are an important bacterial component responsible for bone destruction through the induction of osteoclast differentiation and activation

Bacterial infection can cause inflammatory bone diseases accompanied by the bone destruction resulting from excess generation of osteoclasts. Although lipoproteins are one of the major immunostimulating components of bacteria, little is known about their effects on bone metabolism. In this study, we investigated the role of lipoproteins in bacteria‐induced bone destruction using Staphylococcus aureus wild type, its lipoprotein‐deficient mutant, and synthetic lipopeptides Pam2CSK4 and Pam3CSK4 known to mimic bacterial lipoproteins. Formaldehyde‐inactivated S. aureus or the synthetic lipopeptides induced severe bone loss in the femurs of mice after intraperitoneal administration and in a calvarial bone implantation model, whereas the lipoprotein‐deficient S. aureus did not show such effects. Mechanism studies further identified three action mechanisms for the lipopeptide‐induced osteoclast differentiation and bone resorption via (i) enhancement of osteoclast differentiation through Toll‐like receptor 2 and MyD88‐dependent signaling pathways; (ii) induction of pro‐inflammatory cytokines, TNF‐α and IL‐6; and (iii) upregulation of RANKL expression with downregulation of osteoprotegerin expression in osteoblasts. Taken together, these results suggest that lipoprotein might be an important bacterial component responsible for bone destruction during bacterial infections through augmentation of osteoclast differentiation and activation.

Receptor activator of NF-κB ligand enhances the activity of macrophages as antigen presenting cells

Receptor activator of NFκB ligand (RANKL) is known as a key regulator of osteoclastogenesis. However, the fact that fibroblasts and periodontal ligament cells express RANKL in response to bacterial substances, suggests that RANKL may have evolved as a part of the immunity to infection. As RANKL increases the survival and activity of dendritic cells, it may have similar effects on macrophages. To address this issue, we studied the effect of RANKL on various functions of macrophages using mouse bone marrow derived macrophages. RANKL enhanced the survival of macrophages and up-regulated the expression of CD86. RANKL-treated macrophages showed increased allogeneic T cell activation and phagocytic activity compared to control cells. In addition, RANKL increased the expression of TNFα, MCP-1, and IL-6 but not of IL-10, IL-12, IFN-γ, and iNOS. Collectively, RANKL augmented the activity of macrophages especially as antigen presenting cells, suggesting its new role in immune regulation.

Lipoteichoic acid of Enterococcus faecalis induces the expression of chemokines via TLR2 and PAFR signaling pathways

Enterococcus faecalis is one of the most common opportunistic pathogens responsible for nosocomial infections, and its LTA is known as an important virulence factor causing inflammatory responses. As chemokines play a key role in inflammatory diseases by triggering leukocyte infiltration into the infection site, we purified EfLTA and investigated its effect on the expression of chemokines, IP‐10, MIP‐1α, and MCP‐1, in murine macrophages. EfLTA induced the expression of these chemokines at the mRNA and protein levels. TLR2, CD14, and MyD88 were involved in the EfLTA‐induced chemokine expression, as the expression was reduced remarkably in macrophages derived from TLR2‐, CD14‐, or MyD88‐deficient mice. EfLTA induced phosphorylation of MAPKs and enhanced the DNA‐binding activity of NF‐κB, AP‐1, and NF‐IL6 transcription factors. The induction of IP‐10 required ERK, JNK, p38 MAPK, PKC, PTK, PI3K, and ROS. We noticed that all of these signaling molecules, except p38 MAPK and ROS, were indispensable for the induction of MCP‐1 and MIP‐1α. Interestingly, the EfLTA‐induced chemokine expression was mediated through PAFR/JAK/STAT1 signaling pathways without IFN‐β involvement, which is different from LPS‐induced chemokine expression requiring IFN‐β/JAK/STAT1 signaling pathways. Furthermore, the culture supernatant of EfLTA‐treated RAW 264.7 cells promoted the platelet aggregation, and exogenous PAF induced the chemokine expression in macrophages derived from WT and TLR2‐deficient mice. These results suggest that EfLTA induces the expression of chemokines via signaling pathways requiring TLR2 and PAFR, which is distinct from that of LPS‐induced chemokine expression.

Alpha-amylase is a human salivary protein with affinity to lipopolysaccharide of Aggregatibacter actinomycetemcomitans

Aggregatibacter actinomycetemcomitans lipopolysaccharide (Aa.LPS) is a major virulence factor associated with aggressive periodontitis. Although the recognition of Aa.LPS is potentially initiated by salivary proteins in the oral cavity, Aa.LPS-binding proteins (Aa.LPS-BPs) in saliva are poorly characterized. The purpose of this study was to capture and identify Aa.LPS-BPs in human saliva using a LTQ-Orbitrap hybrid Fourier transform mass spectrometry. Aa.LPS conjugated onto N-hydroxysuccinimidyl-Sepharose(®) 4 Fast Flow beads (Aa.LPS-beads) activated Toll-like receptor 4 and produced nitric oxide and Interferon gamma-inducible protein-10, implying that the conjugation process did not alter the biological properties of Aa.LPS. Aa.LPS-BPs were subsequently isolated from the nine human saliva samples from healthy individuals with the Aa.LPS-beads followed by identification with the mass spectrometry. Aa.LPS-BPs include α-amylase, serum albumin, cystatin, lysozyme C, submaxillary gland androgen-regulated protein 3B, immunoglobulin subunits, polymeric immunoglobulin receptor, deleted in malignant brain tumors 1, prolactin-inducible protein, lipocalin-1, and basic salivary proline-rich protein 2. Specific binding was validated using a pull-down assay with α-amylase which was captured at the highest frequency. Alpha-amylase demonstrated to interfere with the adherence and biofilm formation of A. actinomycetemcomitans. Even heat-inactivated α-amylase showed the interference to the same extent. Conclusively, we identified unique Aa.LPS-BPs that provide useful information to understand bacterial pathogenesis and host innate immunity in the oral cavity.

Functional characterization of a novel FGFR2 mutation, E731K, in craniosynostosis

Craniosynostosis is a condition in which some or all of the sutures in the skull of an infant close prematurely. Fibroblast growth factor receptor 2 (FGFR2) mutations are a well‐known cause of craniosynostosis. Many syndromes that comprise craniosynostosis, such as Apert syndrome, Crouzon syndrome, and Pfeiffer syndrome, have one of the phenotypes that have been reported in FGFR2 mutant patients. FGFRs have been reported in four types (FGFR1–4), and upon binding with FGF ligands, signal transduction occurs inside of cells. Activated FGFR stimulates an osteogenic master transcription factor, Runx2, through the MAP kinase and PKC pathways. We obtained a genetic analysis of six Korean patients who have craniosynostosis as a phenotype. All of the patients had at least one mutation in the FGFR2 gene; five of those mutations have already been reported elsewhere, while one mutation is novel and was hypothesized to lead to Apert syndrome. In this study, we reported and functionally analyzed a novel mutation of the FGFR2 gene found in a craniosynostosis patient, E731K. The mutation is in the 2nd tyrosine kinase domain in the C‐terminal cytoplasmic region of the molecule. The mutation caused an enhanced phosphorylation of the FGFR2E731K and ERK‐MAP kinase, the stimulation of transcriptional activity of Runx2, and consequently, the enhancement of osteogenic marker gene expression. We conclude that the substitution of E731K in FGFR2 is a novel mutation that resulted in a constitutive activation of the receptor and ultimately resulted in premature suture obliteration. J. Cell. Biochem. 113: 457–464, 2012.

Staphylococcus aureus induces IL-8 expression through its lipoproteins in the human intestinal epithelial cell, Caco-2

Staphylococcus aureus can cause the intestinal inflammatory diseases. However, little is known about the molecular mechanism of S. aureus infection in the intestine. In the present study, we investigated whether S. aureus could stimulate human intestinal epithelial cells triggering inflammation. When the human intestinal epithelial cell-line, Caco-2, and the primary colon cells were stimulated with ethanol-inactivated S. aureus, IL-8 expression was induced in a dose-dependent manner. The inactivated S. aureus preferentially stimulated Toll-like receptor (TLR) 2 rather than TLR4. Lipoproteins, lipoteichoic acid (LTA), and peptidoglycan (PGN) are considered as potential TLR2 ligands of S. aureus. Interestingly, S aureus lipoproteins and Pam2CSK4 mimicking Gram-positive bacterial lipoproteins, but not LTA and PGN of S. aureus, significantly induced IL-8 expression in Caco-2 cells. Furthermore, lipoprotein-deficient S. aureus mutant strain failed to induce IL-8 production. Collectively, these results suggest that S. aureus stimulates the human intestinal epithelial cells to induce the chemokine IL-8 production through its lipoproteins, potentially contributing the development of intestinal inflammation.

Enterococcus faecalis lipoteichoic acid suppresses Aggregatibacter actinomycetemcomitans lipopolysaccharide-induced IL-8 expression in human periodontal ligament cells.

Periodontitis is caused by multi-bacterial infection and Aggregatibacter actinomycetemcomitans and Enterococcus faecalis are closely associated with inflammatory periodontal diseases. Although lipopolysaccharide (LPS) of A. actinomycetemcomitans (Aa.LPS) and lipoteichoic acid of E. faecalis (Ef.LTA) are considered to be major virulence factors evoking inflammatory responses, their combinatorial effect on the induction of chemokines has not been investigated. In this study, we investigated the interaction between Aa.LPS and Ef.LTA on IL-8 expression in human periodontal ligament (PDL) cells. Aa.LPS, but not Ef.LTA, substantially induced IL-8 expression at the protein and mRNA levels. Interestingly, Ef.LTA suppressed Aa.LPS-induced IL-8 expression without affecting the binding of Aa.LPS to Toll-like receptor (TLR) 4. Ef.LTA reduced Aa.LPS-induced phosphorylation of mitogen-activated protein kinases, including ERK, JNK and p38 kinase. Furthermore, Ef.LTA inhibited the Aa.LPS-induced transcriptional activities of the activating protein 1, CCAAT/enhancer-binding protein and nuclear factor-kappa B transcription factors, all of which are known to regulate IL-8 gene expression. Ef.LTA augmented the expression of IL-1 receptor-associated kinase-M (IRAK-M), a negative regulator of TLR intracellular signaling pathways, in the presence of Aa.LPS at both the mRNA and protein levels. Small interfering RNA silencing IRAK-M reversed the attenuation of Aa.LPS-induced IL-8 expression by Ef.LTA. Collectively, these results suggest that Ef.LTA down-regulates Aa.LPS-induced IL-8 expression in human PDL cells through up-regulation of the negative regulator IRAK-M.