Keith L. Kirkwood

Functional Cooperation between Interleukin-17 and Tumor Necrosis Factor-α Is Mediated by CCAAT/Enhancer-binding Protein Family Members

Interleukin (IL)-17 is a recently described cytokine involved in the amplification of inflammatory responses and pathologies. A hallmark feature of IL-17 is its ability to induce expression of other cytokines and chemokines. In addition, IL-17 potently synergizes with tumor necrosis factor-α (TNFα) to up-regulate expression of many target genes, particularly IL-6. Despite the many observations of IL-17 signaling synergy observed to date, little is known about the molecular mechanisms that underlie this phenomenon. In the osteoblastic cell line MC-3T3, we have found that IL-17 and TNFα exhibit potent synergy in mediating IL-6 secretion. Here, we show that at least part of the functional cooperation between IL-17 and TNFα occurs at the level of IL-6 gene transcription. Both the NF-κB and CCAAT/enhancer-binding protein (C/EBP; NF-IL6) sites in the IL-6 promoter are important for cooperative gene expression, but NF-κB does not appear to be the direct target of the combined signal. Microarray analysis using the Affymetrix mouse MG-U74v2 chip identified C/EBPδ as another gene target of combined IL-17- and TNFα-induced signaling. Because C/EBP family members are known to control IL-6, we examined whether enhanced C/EBPδ expression is involved in the cooperative up-regulation of IL-6 by IL-17 and TNFα. Accordingly, we show that C/EBPδ (or the related transcription factor C/EBPβ) is essential for expression of IL-6. Moreover, overexpression of C/EBPδ (and, to a lesser extent, C/EBPβ) could substitute for the IL-17 signal at the level of IL-6 transcription. Thus, C/EBP family members, particularly C/EBPδ, appear to be important for the functional cooperation between IL-17 and TNFα.

Cementoblasts Maintain Expression of Osteocalcin in the Presence of Mineral Trioxide Aggregate

This study investigated the effects of mineral trioxide aggregate on cementoblast growth and osteocalcin production in tissue culture. For cellular morphology studies, cementoblasts on mineral trioxide aggregate, IRM, and amalgam were incubated for 48 h then fixed for scanning electron microscopic analysis. For gene expression on mineral trioxide aggregate and IRM, reverse transcriptase polymerase chain reaction was performed using primer sets for glyceraldehyde-3-phosphate dehydrogenase, type I collagen, alkaline phosphatase, osteocalcin, and bone sialoprotein after 3 and 5 days. In vitro matrix protein expression was evaluated by confocal microscopy for the presence of osteocalcin on MTA after 7 and 12 days. Images were compared with controls to assess qualitative differences. Results suggest that mineral trioxide aggregate permits cementoblast attachment and growth and the production of mineralized matrix gene and protein expression. Our data indicates that mineral trioxide aggregate can be considered cementoconductive.

INTERACTIONS BETWEEN EXTRACELLULAR SIGNAL-REGULATED KINASE 1/2 AND P38 MAP KINASE PATHWAYS IN THE CONTROL OF RUNX2 PHOSPHORYLATION AND TRANSCRIPTIONAL ACTIVITY

RUNX2, a key transcription factor for osteoblast differentiation, is regulated by ERK1/2 and p38 MAP kinase‐mediated phosphorylation. However, the specific contribution of each kinase to RUNX2‐dependent transcription is not known. Here we investigate ERK and p38 regulation of RUNX2 using a unique P‐RUNX2‐specific antibody. Both MAP kinases stimulated RUNX2 Ser319 phosphorylation and transcriptional activity. However, a clear preference for ERK1 versus p38α/β was found when the ability of these MAPKs to phosphorylate and activate RUNX2 was compared. Similarly, ERK1 preferentially bound to a consensus MAPK binding site on RUNX2 that was essential for the activity of either kinase. To assess the relative contribution of ERK1/2 and p38 to osteoblast gene expression, MC3T3‐E1 preosteoblast cells were grown in control or ascorbic acid (AA)‐containing medium ± BMP2/7. AA‐induced gene expression, which requires collagen matrix synthesis, was associated with parallel increases in P‐ERK and RUNX2‐S319‐P in the absence of any changes in P‐p38. This response was blocked by ERK, but not p38, inhibition. Significantly, in the presence of AA, BMP2/7 synergistically stimulated RUNX2 S319 phosphorylation and transcriptional activity without affecting total RUNX2 and this response was totally dependent on ERK/MAPK activity. In contrast, although p38 inhibition partially blocked BMP‐dependent transcription, it did not affect RUNX2 S319 phosphorylation, suggesting the involvement of other phosphorylation sites and/or transcription factors in this response. Based on this work, we conclude that extracellular matrix and BMP regulation of RUNX2 phosphorylation and transcriptional activity in osteoblasts is predominantly mediated by ERK rather than p38 MAPKs.

p38 MAPK regulates IL-1β induced IL-6 expression through mRNA stability in osteoblasts

Osteoblast‐derived IL‐6 functions in coupled bone turnover by supporting osteoclastogenesis favoring bone resorption instead of bone deposition. Gene regulation of IL‐6 is complex occurring both at transcription and post‐transcription levels. The focus of this paper is at the level of mRNA stability, which is important in IL‐6 gene regulation. Using the MC3T3‐E1 as an osteoblastic model, IL‐6 secretion was dose dependently decreased by SB203580, a p38 MAPK inhibitor. Steady state IL‐6 mRNA was decreased with SB203580 (2 µM) ca. 85% when stimulated by IL‐1β (1–5 ng/ml). These effects require de novo protein synthesis as they were inhibited by cycloheximide. p38 MAPK had minor effects on proximal IL‐6 promoter activity in reporter gene assays. A more significant effect on IL‐6 mRNA stability was observed in the presence of SB203580. Western blot analysis confirmed that SB203580 inhibited p38 MAP kinase, in response to IL‐1β in a dose dependent manner in MC3T3‐E1 cells. Stably transfected MC3T3‐E1 reporter cell lines (MC6) containing green fluorescent protein (GFP) with the 3′untranslated region of IL‐6 were constructed. Results indicated that IL‐1β, TNFα, LPS but not parathyroid hormone (PTH) could increase GFP expression of these reporter cell lines. Endogenous IL‐6 and reporter gene eGFP‐IL‐6 3′UTR mRNA was regulated by p38 in MC6 cells. In addition, transient transfection of IL‐6 3′UTR reporter cells with immediate upstream MAP kinase kinase‐3 and ‐6 increased GFP expression compared to mock transfected controls. These results indicate that p38 MAPK regulates IL‐1β‐stimulated IL‐6 at a post transcriptional mechanism and one of the primary targets of IL‐6 gene regulation is the 3′UTR of IL‐6.

Potent anti-inflammatory effects of systemically administered curcumin modulate periodontal disease in vivo

BACKGROUND AND OBJECTIVE Curcumin is a plant-derived dietary spice with various biological activities, including anticarcinogenic and anti-inflammatory effects. Its therapeutic applications have been studied in a variety of conditions, including rheumatoid arthritis, colon cancer and depression, but no studies have evaluated the effects of curcumin on periodontal disease in vivo. MATERIAL AND METHODS Experimental periodontal disease was induced in rats by placing cotton ligatures around both lower first molars. Curcumin was given to the rats by the intragastric route daily at two dosages (30 and 100 mg/kg) for 15 d. Control animals received ligatures but only the corn oil vehicle by gavage, and no treatment-negative control animals were included. Bone resorption was assessed by micro-computed tomography, and the inflammatory status was evaluated by stereometric analysis. Both RT-qPCR and ELISA were used to determine the expression of interleukin-6, tumor necrosis factor-α and prostaglandin E(2) synthase in the gingival tissues. Modulation of p38 MAPK and nuclear factor-κB activation were assessed by western blotting. RESULTS Bone resorption was effectively induced in the experimental period, but it was not affected by either dose of curcumin. Curcumin effectively inhibited cytokine gene expression at both the mRNA and the protein level and produced a dose-dependent inhibition of the activation of nuclear factor-κB in the gingival tissues. Activation of p38 MAPK was not inhibited by curcumin. Curcumin-treated animals also presented a marked reduction of the inflammatory cell infiltrate and increased collagen content and fibroblastic cell numbers. CONCLUSION Curcumin did not prevent alveolar bone resorption, but its potent anti-inflammatory effect suggests that it may have a therapeutic potential in periodontal diseases.

A p38α Selective Mitogen-Activated Protein Kinase Inhibitor Prevents Periodontal Bone Loss

In the oral microbial environment, Gram-negative bacterial derived lipopolysaccharide (LPS) can initiate inflammatory bone loss as seen in periodontal diseases. p38 Mitogen-activated protein kinase (MAPK) signaling is critical to inflammatory cytokine and LPS-induced cytokine expression, which may contribute toward periodontal bone loss. The purpose of this proof-of-principle study was to evaluate the ability of an orally active p38α MAPK inhibitor (SD-282) to reduce periopathogenic LPS-induced alveolar bone loss in an experimental rat model. Five groups of Sprague-Dawley rats received one of the following treatments: LPS injected to the palatal gingiva adjacent to the maxillary molars three times per week for 8 weeks, LPS plus two doses of SD-282 (15 or 45 mg/kg) twice daily by oral gavage, or control groups given drug vehicle (1% polyethylene glycol) or SD-282 (45 mg/kg) only. Baseline and 8-week alveolar bone loss was assessed by microcomputed tomography (μCT) and histological examination. LPS induced severe bone loss over this time period, whereas control groups were unchanged from baseline measurements. Both doses of SD-282 showed significant protection from LPS-induced bone loss. Bone area and volumetric analysis of maxillas by μCT indicated significant loss of bone volume with LPS treatment, which was blocked with the p38 inhibitor. Histological examination indicated significantly fewer tartate-resistant acid phosphatase-positive osteoclasts and a significant decrease in interleukin (IL)-6, IL-1β, and tumor necrosis factor α expression in p38 inhibitor-treated groups compared with LPS groups by immunostaining. Results from this in vivo study suggest that orally active p38 MAPK inhibitors can reduce LPS-induced inflammatory cytokine production and osteoclast formation and protect against LPS-stimulated alveolar bone loss.

Rap1GAP Promotes Invasion via Induction of Matrix Metalloproteinase 9 Secretion, Which Is Associated with Poor Survival in Low N-Stage Squamous Cell Carcinoma

The objective of the current study was to investigate the effects of Rap1GAP on invasion and progression of head and neck squamous cell carcinoma (SCC) and the role of matrix metalloproteinase (MMP) 9 and MMP2 in this process. Rap1GAP functions by switching off Rap1, the Ras-like protein that has been associated with carcinogenesis. Previous findings suggest that Rap1GAP acts as a tumor suppressor protein in SCC by delaying the G(1)-S transition of the cell cycle. However, cells transfected with Rap1GAP exhibit a more invasive phenotype than corresponding vector-transfected control cells. MMP2 and MMP9 are enzymes that mediate SCC invasion via degradation of the extracellular matrix. Using SCC cells transfected with empty vector or Rap1GAP, cell invasion and MMP secretion were determined by Matrigel assays and gelatin zymography, respectively. Rap1GAP up-regulated transcription and secretion of MMP2 and MMP9, as assayed by quantitative reverse transcription-PCR and zymography. Furthermore, chemical and RNA interference blockade of MMP2/MMP9 inhibited invasion by Rap1GAP-transfected cells. Immunohistochemical staining of a human oropharyngeal SCC tissue microarray showed that Rap1GAP and MMP9 expression and staining intensity are correlated (P < 0.0001) and that, in early N-stage lesions of SCC, high MMP9 is prognostic of poor disease-specific survival (P < 0.05). Furthermore, Rap1GAP staining is correlated with MMP2 (P < 0.03). MMP2 in combination with N stage has a prognostic effect on time to indication of surgery at primary site. MMP2 intensity is also positively correlated with T stage (P < 0.015). In conclusion, Rap1GAP inhibits tumor growth but induces MMP2- and MMP9-mediated SCC invasion and tumor progression, suggesting a role for this protein as a biomarker for early N-stage, aggressive SCCs.

MAP Kinase Phosphatase-1 Protects against Inflammatory Bone Loss

The mitogen-activated protein (MAP) kinase phosphatase (MKP) family plays an important function in regulating the pro-inflammatory cytokines by deactivating MAP kinases. MKP-1 is essential for the dephosphorylation of p38 MAP kinase that regulates expression of IL-6, TNF-α, and IL-1β. We hypothesized that MKP-1 regulates inflammatory bone loss in experimental periodontitis. Wild-type and Mkp-1−/− mice received A. actinomycetemcomitans LPS injection in the palatal region or PBS control 3 times/wk for 30 days. Mice were killed, and maxillae were assessed by microcomputed tomography, histological analysis, and TRAP staining for measurement of bone loss, extent of inflammation, and degree of osteoclastogenesis. Results indicated that, in LPS-injected Mkp-1−/− mice, significantly greater bone loss occurred with more inflammatory infiltrate and a significant increase in osteoclastogenesis compared with Mkp-1−/− control sites or either wild-type group. Analysis of these data indicates that MKP-1 plays a key role in the regulation of inflammatory bone loss.

Control of Cytokine mRNA Expression by RNA-binding Proteins and microRNAs

Cytokines are critical mediators of inflammation and host defenses. Regulation of cytokines can occur at various stages of gene expression, including transcription, mRNA export, and post- transcriptional and translational levels. Among these modes of regulation, post-transcriptional regulation has been shown to play a vital role in controlling the expression of cytokines by modulating mRNA stability. The stability of cytokine mRNAs, including TNFα, IL-6, and IL-8, has been reported to be altered by the presence of AU-rich elements (AREs) located in the 3′-untranslated regions (3′UTRs) of the mRNAs. Numerous RNA-binding proteins and microRNAs bind to these 3′UTRs to regulate the stability and/or translation of the mRNAs. Thus, this paper describes the cooperative function between RNA-binding proteins and miRNAs and how they regulate AU-rich elements containing cytokine mRNA stability/degradation and translation. These mRNA control mechanisms can potentially influence inflammation as it relates to oral biology, including periodontal diseases and oral pharyngeal cancer progression.

Non-Surgical Chemotherapeutic Treatment Strategies for the Management of Periodontal Diseases

Periodontal diseases are initiated by subgingival periodontal pathogens in susceptible periodontal sites. The host immune response toward periodontal pathogens helps to sustain periodontal disease and eventual alveolar bone loss. Numerous adjunctive therapeutic strategies have evolved to manage periodontal diseases. Systemic and local antibiotics, antiseptics, and past and future host immune modulatory agents are reviewed and discussed to facilitate the dental practitioners appreciation of this ever-growing field in clinical periodontics.