Evaluation of Toll-Like Receptor 2 and 4 RNA Expression and the Cytokine Profile in Postmenopausal Women with Metabolic Syndrome

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

Short-term effects of triiodothyronine on thyroid hormone receptor alpha by PI3K pathway in adipocytes, 3T3-L1

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

Cardiac dysfunction induced by obesity es not related to beta-adrenergic system impairment at the receptor-signalling pathway

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

Down-regulation of protease activated receptor 2, interleukin-17 and other pro-inflammatory genes by subantimicrobial doxycycline dose in a rat periodontitis model

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

PPAR gamma activation attenuates cold-induced upregulation of thyroid status and brown adipose tissue PGC-1 alpha and D2

Festuccia WT, Blanchard PG, Oliveira TB, Magdalon J, Paschoal VA, Richard D, Deshaies Y. PPAR gamma activation attenuates cold-induced upregulation of thyroid status and brown adipose tissue PGC-1 alpha and D2. Am J Physiol Regul Integr Comp Physiol 303: R1277-R1285, 2012. First published October 24, 2012; doi:10.1152/ajpregu.00299.2012.-Here, we investigated whether pharmacological PPAR gamma activation modulates key early events in brown adipose tissue (BAT) recruitment induced by acute cold exposure with the aim of unraveling the interrelationships between sympathetic and PPAR gamma signaling. Sprague-Dawley rats treated or not with the PPAR gamma ligand rosiglitazone (15 mg.kg(-1).day(-1), 7 days) were kept at 23 degrees C or exposed to cold (5 degrees C) for 24 h and evaluated for BAT gene expression, sympathetic activity, thyroid status, and adrenergic signaling. Rosiglitazone did not affect the reduction in body weight gain and the increase in feed efficiency, VO2, and BAT sympathetic activity induced by 24-h cold exposure. Rosiglitazone strongly attenuated the increase in serum total and free T4 and T3 levels and BAT iodothyronine deiodinase type 2 (D2) and PGC-1 alpha mRNA levels and potentiated the reduction in BAT thyroid hormone receptor (THR) beta mRNA levels induced by cold. Administration of T3 to rosiglitazone-treated rats exacerbated the cold-induced increase in energy expenditure but did not restore a proper activation of D2 and PGC-1 alpha, nor further increased uncoupling protein 1 expression. Regarding adrenergic signaling, rosiglitazone did not affect the changes in BAT cAMP content and PKA activity induced by cold. Rosiglitazone alone or in combination with cold increased CREB binding to DNA, but it markedly reduced the expression of one of its major coactivators, CREB binding protein. In conclusion, pharmacological PPAR gamma activation impairs short-term cold elicitation of BAT adrenergic and thyroid signaling, which may result in abnormal tissue recruitment and thermogenic activity.

Human beta-defensin 2 and protease activated receptor-2 expression in patients with chronic periodontitis

Objective: Some previous studies have shown that gingipains, trypsin-like proteases produced by Porphyromonas gingivalis, up-regulate human beta defensin-2 (HBD-2) mRNA expression through protease-activated receptor-2 (PAR(2)) in gingival epithelial cells. This study aimed at investigating salivary HBD-2 levels and crevicular PAR(2) mRNA expression in human chronic periodontitis and evaluating whether periodontal treatment affected this process. Methods: Salivary and gingival crevicular fluid (GCF) samples were collected from periodontally healthy (control) and chronic periodontitis patients at baseline and 50 days after nonsurgical periodontal treatment. Salivary HBD-2, and GCF TNF-alpha levels were analysed by ELISA, and PAR(2) mRNA at the GCF was evaluated by RT-PCR. Results: P. gingivalis was significantly (p < 0.05) more prevalent in patients with chronic periodontitis when compared to controls. This prevalence decreased after periodontal therapy (p < 0.0001). The control group showed statistically significant lower levels of HBD-2, TNF-alpha, and PAR(2) expression when compared to the chronic periodontitis group. In addition, periodontal treatment significantly reduced PAR(2) expression and HBD-2 levels in chronic periodontitis patients (p < 0.001). Conclusions: Our results suggest that salivary HBD-2 levels and PAR(2) mRNA expression from GCF are higher in subjects with chronic periodontitis than in healthy subjects, and that periodontal treatment decreases both HBD-2 levels and PAR(2) expression. (C) 2012 Elsevier Ltd. All rights reserved.

Preservation of Alpha-3 Neuronal Nicotinic Acetylcholine Receptor Expression in Sympathetic Ganglia After Brain Death

The goal of this study was to evaluate if the immunohistochemical expression of alpha-3 neuronal nicotinic acetylcholine receptor subunit in sympathetic ganglia remains stable after brain death, determining the possible use of sympathetic thoracic ganglia from subjects after brain death as study group. The third left sympathetic ganglion was resected from patients divided in two groups: BD-organ donors after brain death and CON-patients submitted to sympathectomy for hyperhidrosis (control group). Immunohistochemical staining for alpha-3 neuronal nicotinic acetylcholine receptor subunit was performed; strong and weak expression areas were quantified in both groups. The BD group showed strong alpha-3 neuronal nicotinic acetylcholine receptor expression in 6.55% of the total area, whereas the CON group showed strong expression in 5.91% (p = 0.78). Weak expression was found in 6.47% of brain-dead subjects and in 7.23% of control subjects (p = 0.31). Brain death did not affect the results of the immunohistochemical analysis of sympathetic ganglia, and its use as study group is feasible.

Toll-like Receptor 2 Knockout Mice Showed Increased Periapical Lesion Size and Osteoclast Number

Introduction: The aim of this study was to characterize the formation and progression of experimentally induced periapical lesions in TLR2 knockout (TLR2 KO) mice. Methods: Periapical lesions were induced in molars of 28 wild type (WT) and 27 TLR2 KO mice. After 7, 21, and 42 days, the animals were euthanized, and the mandibles were subjected to histotechnical processing. Hematoxylin-eosin-stained sections were examined under conventional light microscopy for the description of pulpal, apical, and periapical features and under fluorescence microscopy for the determination of the periapical lesion size. The subsequent sections were evaluated by tartrate resistant acid phosphatase histoenzymology (osteoclasts), Brown and Brenn staining (bacteria), and immunohistochemistry (RANK, RANKL, and OPG). Data were analyzed by the Mann-Whitney U and Kruskal-Wallis tests (alpha = 0.05), Results: The WT group showed significant differences (P < .05) in the periapical lesion size and the osteoclast number between 7 and 42 days and between 21 and 42 days. In the TLR2 KO group, significant differences (P < .05) in the periapical lesion size and the osteoclast number were found between 7 days and the other periods. There was a significant difference (P < .05) between the 2 types of animal regarding the periapical lesion size, which was larger in the TLR2 KO animals. No significant differences (P > .05) were found between WT and TLR2 KO mice related to the pulpal, apical, and periapical features; bacteria localization; and immunohistochemical results (except for RANK expression). Conclusions: TLR2 KO animals developed larger periapical lesions with a greater number of osteoclasts, indicating the important role of this receptor in the host's immune and inflammatory response to root canal and periradicular infection. (J Endod 2012;38:803-813)

Real-time PCR quantitation of glucocorticoid receptor alpha isoform

Abstract Background The expression of glucocorticoid-receptor (GR) seems to be a key mechanism in the regulation of glucocorticoid (GC) sensitivity and is potentially involved in cases of GC resistance or hypersensitivity. The aim of this study is to describe a method for quantitation of GR alpha isoform (GRα) expression using real-time PCR (qrt-PCR) with analytical capabilities to monitor patients, offering standard-curve reproducibility as well as intra- and inter-assay precision. Results Standard-curves were constructed by employing standardized Jurkat cell culture procedures, both for GRα and BCR (breakpoint cluster region), as a normalizing gene. We evaluated standard-curves using five different sets of cell culture passages, RNA extraction, reverse transcription, and qrt-PCR quantification. Intra-assay precision was evaluated using 12 replicates of each gene, for 2 patients, in a single experiment. Inter-assay precision was evaluated on 8 experiments, using duplicate tests of each gene for two patients. Standard-curves were reproducible, with CV (coefficient of variation) of less than 11%, and Pearson correlation coefficients above 0,990 for most comparisons. Intra-assay and inter-assay were 2% and 7%, respectively. Conclusion This is the first method for quantitation of GRα expression with technical characteristics that permit patient monitoring, in a fast, simple and robust way.

Periodontal treatment downregulates protease-activated receptor 2 in human gingival crevicular fluid cells

Protease-activated receptor 2 (PAR2) is implicated in the pathogenesis of chronic inflammatory diseases, including periodontitis; it can be activated by gingipain and produced by Porphyromonas gingivalis and by neutrophil protease 3 (P3). PAR2 activation plays a relevant role in inflammatory processes by inducing the release of important inflammatory mediators associated with periodontal breakdown. The effects of periodontal treatment on PAR2 expression and its association with levels of proinflammatory mediators and activating proteases were investigated in chronic periodontitis patients. Positive staining for PAR2 was observed in gingival crevicular fluid cells and was reflective of tissue destruction. Overexpression of PAR2 was positively associated with inflammatory clinical parameters and with the levels of interleukin-6 (IL-6), IL-8, tumor necrosis factor alpha, matrix metalloprotease 2 (MMP-2), MMP-8, hepatocyte growth factor, and vascular endothelial growth factor. Elevated levels of gingipain and P3 and decreased levels of dentilisin and the protease inhibitors secretory leukocyte protease inhibitor and elafin were also associated with PAR2 overexpression. Healthy periodontal sites from individuals with chronic periodontitis showed diminished expression of PAR2 mRNA and the PAR2 protein (P < 0.05). Furthermore, periodontal treatment resulted in decreased PAR2 expression and correlated with decreased expression of inflammatory mediators and activating proteases. We concluded that periodontal treatment resulted in decreased levels of proteases and that proinflammatory mediators are associated with decreased PAR2 expression, suggesting that PAR2 expression is influenced by the presence of periodontal infection and is not a constitutive characteristic favoring periodontal inflammation.

Cardiac G-protein-coupled receptor kinase 2 ablation induces a novel Ca2+ handling phenotype resistant to adverse alterations and remodeling after myocardial infarction

G-protein-coupled receptor kinase 2 (GRK2) is a primary regulator of β-adrenergic signaling in the heart. G-protein-coupled receptor kinase 2 ablation impedes heart failure development, but elucidation of the cellular mechanisms has not been achieved, and such elucidation is the aim of this study.

Insulin resistance in mice lacking neuronal nitric oxide synthase is related to an alpha-adrenergic mechanism

BACKGROUND: nitric oxide (NO) plays an important role in the regulation of cardiovascular and glucose homeostasis. Mice lacking the gene encoding the neuronal isoform of nitric oxide synthase (nNOS) are insulin-resistant, but the underlying mechanism is unknown. nNOS is expressed in skeletal muscle tissue where it may regulate glucose uptake. Alternatively, nNOS driven NO synthesis may facilitate skeletal muscle perfusion and substrate delivery. Finally, nNOS dependent NO in the central nervous system may facilitate glucose disposal by decreasing sympathetic nerve activity. METHODS: in nNOS null and control mice, we studied whole body glucose uptake and skeletal muscle blood flow during hyperinsulinaemic clamp studies in vivo and glucose uptake in skeletal muscle preparations in vitro. We also examined the effects of alpha-adrenergic blockade (phentolamine) on glucose uptake during the clamp studies. RESULTS: as expected, the glucose infusion rate during clamping was roughly 15 percent lower in nNOS null than in control mice (89 (17) vs 101 (12) [-22 to -2]). Insulin stimulation of muscle blood flow in vivo, and intrinsic muscle glucose uptake in vitro, were comparable in the two groups. Phentolamine, which had no effect in the wild-type mice, normalised the insulin sensitivity in the mice lacking the nNOS gene. CONCLUSIONS: insulin resistance in nNOS null mice was not related to defective insulin stimulation of skeletal muscle perfusion and substrate delivery or insulin signaling in the skeletal muscle cell, but to a sympathetic alpha-adrenergic mechanism.

The Protein-tyrosine-phosphatase SHP2 is phosphorylated on serine residues 576 and 591 by protein kinase C isoforms alpha, beta 1, beta 2, and eta

To study whether protein kinase C (PKC) isoforms can interact with protein-tyrosine-phosphatases (PTPs) which are connected to the insulin signaling pathway, we co-overexpressed PKC isoforms together with insulin receptor, docking proteins, and the PTPs SHP1 and SHP2 in human embryonic kidney (HEK) 293 cells. After phorbol ester induced activation of PKC isoforms alpha, beta 1, beta 2, and eta, we could show a defined gel mobility shift of SHP2, indicating phosphorylation on serine/threonine residues. This phosphorylation was not dependent on insulin receptor or insulin receptor substrate-1 (IRS-1) overexpression and did not occur for the closely related phosphatase SHP1. Furthermore, PKC phosphorylation of SHP2 was completely blocked by the PKC inhibitor bisindolylmaleimide and was not detectable when SHP2 was co-overexpressed with kinase negative mutants of PKC beta 1 and -beta 2. The phosphorylation also occurred on endogenous SHP2 in Chinese hamster ovary (CHO) cells stably overexpressing PKC beta 2. Using point mutants of SHP2, we identified serine residues 576 and 591 as phosphorylation sites for PKC. However, no change of phosphatase activity by TPA treatment was detected in an in vitro assay. In summary, SHP2 is phosphorylated on serine residues 576 and 591 by PKC isoforms alpha, beta 1, beta 2, and eta.

Tenascin-W is found in malignant mammary tumors, promotes alpha8 integrin-dependent motility and requires p38MAPK activity for BMP-2 and TNF-alpha induced expression in vitro

Tenascins represent a family of extracellular matrix glycoproteins with distinctive expression patterns. Here we have analyzed the most recently described member, tenascin-W, in breast cancer. Mammary tumors isolated from transgenic mice expressing hormone-induced oncogenes reveal tenascin-W in the stroma around lesions with a high likelihood of metastasis. The presence of tenascin-W was correlated with the expression of its putative receptor, alpha8 integrin. HC11 cells derived from normal mammary epithelium do not express alpha8 integrin and fail to cross tenascin-W-coated filters. However, 4T1 mammary carcinoma cells do express alpha8 integrin and their migration is stimulated by tenascin-W. The expression of tenascin-W is induced by BMP-2 but not by TGF-beta1, though the latter is a potent inducer of tenascin-C. The expression of tenascin-W is dependent on p38MAPK and JNK signaling pathways. Since preinflammatory cytokines also act through p38MAPK and JNK signaling pathways, the possible role of TNF-alpha in tenascin-W expression was also examined. TNF-alpha induced the expression of both tenascin-W and tenascin-C, and this induction was p38MAPK- and cyclooxygenase-dependent. Our results show that tenascin-W may be a useful diagnostic marker for breast malignancies, and that the induction of tenascin-W in the tumor stroma may contribute to the invasive behavior of tumor cells.

Hypoxia inducible factor-1 alpha induction by tumour necrosis factor-alpha, but not by toll-like receptor agonists, modulates cellular respiration in cultured human hepatocytes

BACKGROUND/AIMS: Genes encoding for some of the mitochondrial proteins are under the control of the transcriptional factor hypoxia inducible factor-1 alpha (HIF-1 alpha), which can accumulate under normoxic conditions in inflammatory states. The aim of this study was to evaluate the effects of cobalt chloride (CoCl(2), a hypoxia mimicking agent), tumour necrosis factor-alpha (TNF-alpha) and toll-like receptor (TLR) -2, -3 and -4 agonists on HIF-1 alpha accumulation, and further on HIF-1 alpha-mediated modulation of mitochondrial respiration in cultured human hepatocytes. METHODS: The human hepatoma cell line HepG2 was used in this study. Cells were treated with CoCl(2), TNF-alpha and TLR-2, -3 and -4 agonists. HIF-1 alpha was determined by Western blotting and mitochondrial respiration in stimulated cells by high-resolution respirometry. RESULTS: CoCl(2), TNF-alpha and TLR agonists induced the expression of HIF-1 alpha in a time-dependent fashion. TNF-alpha and CoCl(2), but not TLR agonists, induced a reduction in complex I-, II- and IV-dependent mitochondrial oxygen consumption. TNF-alpha-associated reduction of cellular oxygen consumption was abolished through inhibition of HIF-1 alpha activity by chetomin (CTM). Pretreatment with cyclosporine A prevented CoCl(2)-induced reduction of complex I- and II-dependent mitochondrial oxygen consumption and TNF-alpha-induced reduction of complex-I-dependent respiration, implicating the involvement of the mitochondrial permeability transition pore openings. TNF-alpha and TLR-2, -3 and -4 agonists induced the expression of vascular endothelial growth factor, which was partially abolished by the blockage of HIF-1 alpha with CTM. CONCLUSIONS: The data suggest that HIF-1 alpha modulates mitochondrial respiration during CoCl(2) and TNF-alpha stimulation, whereas it has no effect when induced with TLR-2, -3 and -4 agonists.