MKK3/6-p38 MAPK signaling is required for IL-1 beta and TNF-alpha-induced RANKL expression in bone marrow stromal cells

Coupled bone turnover is directed by the expression of receptor-activated NF-kappa B ligand (RANKL) and its decoy receptor, osteoprotegerin (OPG). Proinflammatory cytokines, such as interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF-alpha) induce RANKL expression in bone marrow stromal cells. Here, we report that IL-1 beta and TNF-alpha-induced RANKL requires p38 mitogen-activating protein kinase (MAPK) pathway activation for maximal expression. Real-time PCR was used to assess the p38 contribution toward IL-1 beta and TNF-alpha-induced RANKL mRNA expression. Steady-state RANKL RNA levels were increased approximately 17-fold by IL-1 beta treatment and subsequently reduced similar to 70%-90% when p38 MAPK was inhibited with SB203580. RANKL mRNA stability data indicated that p38 MAPK did not alter the rate of mRNA decay in IL-1 beta-induced cells. Using a RANKL-luciferase cell line receptor containing a 120-kB segment of the 5' flanking region of the RANKL gene, reporter expression was stimulated 4-5-fold by IL-1 beta or TNF-alpha treatment. IL-1 beta-induced RANKL reporter expression was completely blocked with specific p38 inhibitors as well as dominant negative mutant constructs of MAPK kinase-3 and -6. In addition, blocking p38 signaling in bone marrow stromal cells partially inhibited IL-1 beta and TNF-alpha-induced osteoclastogenesis in vitro. Results from these studies indicate that p38 MAPK is a major signaling pathway involved in IL-1 beta and TNF-alpha-induced RANKL expression in bone marrow stromal cells.

Lemongrass and citral effect on cytokines production by murine macrophages

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

TNF-alpha acts in the hypothalamus inhibiting food intake and increasing the respiratory quotient - Effects on leptin and insulin signaling pathways

Acting in the hypothalamus, tumor necrosis factor-alpha (TNF-alpha) produces a potent anorexigenic effect. However, the molecular mechanisms involved in this phenomenon are poorly characterized. In this study, we investigate the capacity of TNF-alpha to activate signal transduction in the hypothalamus through elements of the pathways employed by the anorexigenic hormones insulin and leptin. High dose TNF-a promotes a reduction of 25% in 12 h food intake, which is an inhibitory effect that is marginally inferior to that produced by insulin and leptin. In addition, high dose TNF-a increases body temperature and respiratory quotient, effects not reproduced by insulin or leptin. TNF-alpha, predominantly at the high dose, is also capable of activating canonical pro-inflammatory signal transduction in the hypothalamus, inducing JNK, p38, and NF kappa B, which results in the transcription of early responsive genes and expression of proteins of the SOCS family. Also, TNF-a activates signal transduction through JAK-2 and STAT-3, but does not activate signal transduction. through early and intermediary elements of the insulin/leptin signaling pathways such as IRS-2, Akt, ERK and FOXO1. When co-injected with insulin or leptin, TNF-a, at both high and low doses, partially impairs signal transduction through IRS-2, Akt, ERK and FOXO1 but not through JAK-2 and STAT-3. This effect is accompanied by the partial inhibition of the anorexigenic effects of insulin and leptin, when the low, but not the high dose of TNF-alpha is employed. In conclusion, TNF-alpha, on a dose-dependent way, modulates insulin and leptin signaling and action in the hypothalamus. (c) Published by Elsevier B.V.

Plant derived alkaloid (-)-cassine induces anti-inflammatory and anti-hyperalgesics effects in both acute and chronic inflammatory and neuropathic pain models

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

The importance of hormone receptor analysis in osteosarcoma cells growth submitted to treatment with estrogen in association with thyroid hormone

Bone tumor incidence in women peaks at age 50-60, coinciding with the menopause. That estrogen (E2) and triiodothyronine (T3) interact in bone metabolism has been well established. However, few data on the action of these hormones are available. Our purpose was to determine the role of E2 and T3 in the expression of bone activity markers, namely alkaline phosphatase (AP) and receptor activator of nuclear factor κB ligand (RANKL). Two osteosarcoma cell lines: MG-63 (which has both estrogen (ER) and thyroid hormone (TR) receptors) and SaOs-29 (ER receptors only) were treated with infraphysiological E2 associated with T3 at infraphysiological, physiological, and supraphysiological concentrations. Real-time RT-PCR was used for expression analysis. Our results show that, in MG-63 cells, infraphysiological E2 associated with supraphysiological T3 increases AP expression and decreases RANKL expression, while infraphysiological E2 associated with either physiological or supraphysiological T3 decreases both AP and RANKL expression. On the other hand, in SaOs-2 cells, the same hormone combinations had no significant effect on the markers' expression. Thus, the analysis of hormone receptors was shown to be crucial for the assessment of tumor potential growth in the face of hormonal changes. Special care should be provided to patients with T3 and E2 hormone receptors that may increase tumor growth. Copyright © 2007 John Wiley & Sons, Ltd.

Increased apoptosis in osteoclasts and decreased RANKL immunoexpression in periodontium of cimetidine-treated rats

It has been demonstrated that histamine interferes with the recruitment, formation and activity of osteoclasts via H1- and H2-receptors. Cimetidine is a H2-receptor antagonist used for treatment of gastric ulcers that seems to prevent bone resorption. In this study, a possible cimetidine interference was investigated in the number of alveolar bone osteoclasts. The incidence of osteoclast apoptosis and immunoexpression of RANKL (receptor activator of nuclear factor κB ligand) was also evaluated. Adult male rats were treated with 100mg kg-1 of cimetidine for 50days (CimG); the sham group (SG) received saline. Maxillary fragments containing the first molars and alveolar bone were fixed, decalcified and embedded in paraffin. The sections were stained by H&E or submitted to tartrate-resistant acid phosphatase (TRAP) method. TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling) method and immunohistochemical reactions for detecting caspase-3 and RANKL were performed. The number of TRAP-positive osteoclasts, the frequency of apoptotic osteoclasts and the numerical density of RANKL-positive cells were obtained. Osteoclast death by apoptosis was confirmed by transmission electron microscopy (TEM). In CimG, TRAP-positive osteoclasts with TUNEL-positive nuclei and caspase-3-immunolabeled osteoclasts were found. A significant reduction in the number of TRAP-positive osteoclasts and a high frequency of apoptotic osteoclasts were observed in CimG. Under TEM, detached osteoclasts from the bone surface showed typical features of apoptosis. Moreover, a significant reduction in the numerical density of RANKL-positive cells was observed in CimG. The significant reduction in the number of osteoclasts may be due to cimetidine-induced osteoclast apoptosis. However, RANKL immunoexpression reduction also suggests a possible interference of cimetidine treatment in the osteoclastogenesis. © 2012 The Authors Journal of Anatomy © 2012 Anatomical Society.

Guided implant surgery: What is the influence of this new technique on bone cell viability?

Purpose: To evaluate the effect of implant osteotomy on immediate bone cell viability, comparing guided surgery for implant placement with the classic drilling procedure. Materials and Methods: For this study, 20 rabbits were used. The animals were divided into a guided surgery group (GG) and a control group (CG) and were then divided into 4 subgroups - subgroups 1, 2, 3, and 4 - corresponding to drills used 10, 20, 30, and 40 times, respectively. All animals received 5 osteotomies in each tibia, by use of the classic drilling procedure in one tibia and guided surgery in the other tibia. The osteotomized areas were removed and processed immunohistochemically for detection of osteocalcin, receptor activator of nuclear factor κB ligand (RANKL), osteoprotegerin (OPG), and caspase 3. Results: Immunohistochemical analysis showed that osteocalcin expression was initially higher in the CG and remained constant after drill reutilization. Although the expressions of RANKL and OPG were not statistically different for the GG and CG, the RANKL/OPG ratio tended to be higher for the GG. Moreover, caspase 3 expression was elevated in the GG, proportionally to the number of osteotomies, indicating an increase in the apoptosis index in the GG. Conclusions: The classic drilling procedure is more favorable to cell viability than guided surgery.© 2013 American Association of Oral and Maxillofacial Surgeons.

Anti-inflammatory mechanisms of the annexin A1 protein and its mimetic peptide Ac2-26 in models of ocular inflammation in vivo and in vitro

Annexin A1 (AnxA1) is a protein that displays potent anti-inflammatory properties, but its expression in eye tissue and its role in ocular inflammatory diseases have not been well studied. We investigated the mechanism of action and potential uses of AnxA1 and its mimetic peptide (Ac2-26) in the endotoxin-induced uveitis (EIU) rodent model and in human ARPE-19 cells activated by LPS. In rats, analysis of untreated EIU after 24 and 48 h or EIU treated with topical applications or with a single s.c. injection of Ac2-26 revealed the anti-inflammatory actions of Ac2-26 on leukocyte infiltration and on the release of inflammatory mediators; the systemic administration of Boc2, a formylated peptide receptor (fpr) antagonist, abrogated the peptide's protective effects. Moreover, AnxA1-/- mice exhibited exacerbated EIU compared with wild-type animals. Immunohistochemical studies of ocular tissue showed a specific AnxA1 posttranslational modification in EIU and indicated that the fpr2 receptor mediated the anti-inflammatory actions of AnxA1. In vitro studies confirmed the roles of AnxA1 and fpr2 and the protective effects of Ac2-26 on the release of chemical mediators in ARPE-19 cells. Molecular analysis of NF-κB translocation and IL-6, IL-8, and cyclooxygenase-2 gene expression indicated that the protective effects of AnxA1 occur independently of the NF-κB signaling pathway and possibly in a posttranscriptional manner. Together, our data highlight the role of AnxA1 in ocular inflammation, especially uveitis, and suggest the use of AnxA1 or its mimetic peptide Ac2-26 as a therapeutic approach. Copyright © 2013 by The American Association of Immunologists, Inc.

The role of cytokines in inflammatory bone loss

Chronic inflammatory processes close to bone often lead to loss of bone in diseases such as rheumatoid arthritis, periodontitis, loosened joint prosthesis and tooth implants. This is mainly due to local formation of bone resorbing osteoclasts which degrade bone without any subsequent coupling to new bone formation. Crucial for osteoclastogenesis is stimulation of mononuclear osteoclast progenitors by macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor-κB ligand (RANKL) which induces their differentiation along the osteoclastic lineage and the fusion to mature, multinucleated osteoclasts. M-CSF and RANKL are produced by osteoblasts/ osteocytes and by synovial and periodontal fibroblasts and the expression is regulated by pro- and anti-inflammatory cytokines. These cytokines also regulate osteoclastic differentiation by direct effects on the progenitor cells. In the present overview, we introduce the basic concepts of osteoclast progenitor cell differentiation and summarize the current knowledge on cytokines stimulating and inhibiting osteoclastogenesis by direct and indirect mechanisms. © Informa Healthcare USA, Inc.

Papel das Yops de yersinia pseudotuberculosis na modulação da resposta imune celular durante infecção experimental

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Influência da ovariectomia associada à dieta deficiente em cálcio na periodontite experimental

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Determinação da ativação de monócitos e de biomarcadores celulares em gestantes portadoras de pré-eclâmpsia

Pós-graduação em Ginecologia, Obstetrícia e Mastologia - FMB

Effect of Qualea multiflora on Macrophages and Tumour Cells

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Curcurnin abrogates LPS-induced pro-inflammatory cytokines in RAW 264.7 macrophages. Evidence for novel mechanisms involving SOCS-1,-3 and p38 MAPK

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Aerobic Exercise Training Prevents Heart Failure-Induced Skeletal Muscle Atrophy by Anti-Catabolic, but Not Anabolic Actions

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)