Role of alpha 7 Nicotinic Acetylcholine Receptor in Calcium Signaling Induced by Prion Protein Interaction with Stress-inducible Protein 1

The prion protein (PrP(C)) is a conserved glycosylphosphatidyl-inositol-anchored cell surface protein expressed by neurons and other cells. Stress-inducible protein 1 (STI1) binds PrP(C) extracellularly, and this activated signaling complex promotes neuronal differentiation and neuroprotection via the extracellular signal-regulated kinase 1 and 2 (ERK1/2) and cAMP-dependent protein kinase 1 (PKA) pathways. However, the mechanism by which the PrPC-STI1 interaction transduces extracellular signals to the intracellular environment is unknown. We found that in hippocampal neurons, STI1-PrP(C) engagement induces an increase in intracellular Ca(2+) levels. This effect was not detected in PrP(C)-null neurons or wild-type neurons treated with an STI1 mutant unable to bind PrP(C). Using a best candidate approach to test for potential channels involved in Ca(2+) influx evoked by STI1-PrP(C), we found that alpha-bungarotoxin, a specific inhibitor for alpha 7 nicotinic acetylcholine receptor (alpha 7nAChR), was able to block PrP(C)-STI1-mediated signaling, neuroprotection, and neuritogenesis. Importantly, when alpha 7nAChR was transfected into HEK 293 cells, it formed a functional complex with PrP(C) and allowed reconstitution of signaling by PrP(C)-STI1 interaction. These results indicate that STI1 can interact with the PrP(C).alpha 7nAChR complex to promote signaling and provide a novel potential target for modulation of the effects of prion protein in neurodegenerative diseases.

The plasmodium receptor for activated C kinase protein inhibits Ca(2+) signaling in mammalian cells

Plasmodium falciparum, the most lethal malarial parasite, expresses an ortholog for the protein kinase C (PKC) activator RACK1. However, PKC has not been identified in this parasite, and the mammalian RACK1 can interact with the inositol 1,4,5-trisphosphate receptor (InsP3R). Therefore we investigated whether the Plasmodium ortholog PfRACK also can affect InsP3R-mediated Ca(2+) signaling in mammalian cells. GFP-tagged PfRACK and endogenous RACK1 were expressed in a similar distribution within cells. PfRACK inhibited agonist-induced Ca(2+) signals in cells expressing each isoform of the InsP3R, and this effect persisted when expression of endogenous RACK1 was reduced by siRNA. PfRACK also inhibited Ca(2+) signals induced by photorelease of caged InsP3. These findings provide evidence that PfRACK directly inhibits InsP3-mediated Ca(2+) signaling in mammalian cells. Interference with host cell signaling pathways to subvert the host intracellular milieu may be an important mechanism for parasite survival. (C) 2009 Elsevier Inc. All rights reserved.

5-HT(1B) receptor in the suprachiasmatic nucleus of the common marmoset (Callithrix jacchus)

Serotonin (5-HT) is involved in the fine adjustments at several brain centers including the core of the mammal circadian timing system (CTS) and the hypothalamic suprachiasmatic nucleus (SCN). The SCN receives massive serotonergic projections from the midbrain raphe nuclei, whose inputs are described in rats as ramifying at its ventral portion overlapping the retinohypothalamic and geniculohypothalamic fibers. In the SCN, the 5-HT actions are reported as being primarily mediated by the 5-HT1 type receptor with noted emphasis for 5-HT(1B) subtype, supposedly modulating the retinal input in a presynaptic way. In this study in a New World primate species, the common marmoset (Callithrix jacchus), we showed the 5-HT(1B) receptor distribution at the dorsal SCN concurrent with a distinctive location of 5-HT-immunoreactive fibers. This finding addresses to a new discussion on the regulation and synchronization of the circadian rhythms in recent primates. (C) 2010 Elsevier Ireland Ltd. All rights reserved.

Increased pancreatic islet mass is accompanied by activation of the insulin receptor substrate-2/serine-threonine kinase pathway and augmented cyclin D(2) protein levels in insulin-resistant rats

It is well known that glucocorticoids induce peripheral insulin resistance in rodents and humans. Here, we investigated the structural and ultrastructural modifications, as well as the proteins involved in beta-cell function and proliferation, in islets from insulin-resistant rats. Adult male Wistar rats were made insulin resistant by daily administration of dexamethasone (DEX; 1mg/kg, i.p.) for five consecutive days, whilst control (CTL) rats received saline alone. Structure analyses showed a marked hypertrophy of DEX islets with an increase of 1.7-fold in islet mass and of 1.6-fold in islet density compared with CTL islets (P < 0.05). Ultrastructural evaluation of islets revealed an increased amount of secreting organelles, such as endoplasmic reticulum and Golgi apparatus in DEX islets. Mitotic figures were observed in DEX islets at structural and ultrastructural levels. Beta-cell proliferation, evaluated at the immunohistochemical level using anti-PCNA (proliferating cell nuclear antigen), showed an increase in pancreatic beta-cell proliferation of 6.4-fold in DEX islets compared with CTL islets (P < 0.0001). Increases in insulin receptor substrate-2 (IRS-2), phosphorylated-serine-threonine kinase AKT (p-AKT), cyclin D(2) and a decrease in retinoblastoma protein (pRb) levels were observed in DEX islets compared with CTL islets (P < 0.05). Therefore, during the development of insulin resistance, the endocrine pancreas adapts itself increasing beta-cell mass and proliferation, resulting in an amelioration of the functions. The potential mechanisms that underlie these events involve the activation of the IRS-2/AKT pathway and activation of the cell cycle, mediated by cyclin D(2). These adaptations permit the maintenance of glycaemia at near-physiological ranges.

Absence of TGF-beta RII predicts bone and lung metastasis and is associated with poor prognosis in stage III breast tumors

In the case of operated breast cancer (BC), prognostic markers help to determine if the patient needs additional treatment and predictive markers help the clinician to decide which treatment to use. Thus, a better knowledge of known predictive and prognostic markers and the identification of new markers, may improve the treatment of BC patients. The transforming growth factor-beta type II receptor (TGF-beta RII), a main receptor of transforming growth factor beta pathway, is a potential new prognostic marker. The aims of the present study were to investigate both the predictive and prognostic impact of TGF-beta RII in BC samples. TGF-beta RII protein expression was evaluated using immunohistochemistry on a tissue microarray containing 110 TNM stage III BC samples obtained prior to doxorubicin-based neoadjuvant chemotherapy (NAC). Our results demonstrate that TGF-beta RII did not predict the response to NAC. on the other hand, an association between TGF-beta RII-negative tumor and higher risk of metastasis to lungs and bones was verified. TGF-beta RII negativity was an independent prognostic factor for decreased disease-free and overall survival.

Evaluation of immediate bone-cell viability and of drill wear after implant osteotomies: Immunohistochemistry and scanning electron microscopy analysis

Purpose: This study sought to evaluate the effect of repeated implant drilling on the immediate bone-cell viability, and to evaluate drill wear by scanning electron microscopy.Materials and Methods: The tibiae of 10 rabbits were used, divided into 5 groups (G): G1 corresponded to new drills, and G2, G3, G4, and G5 corresponded to drills used 10, 20, 30, and 40 times, respectively. The animals received 10 sequential osteotomies in each tibia. The animals were euthanized immediately after the osteotomies by perfusion with 4% formaldehyde. Samples then underwent immunohistochemistry processing for ordinal qualitative analysis of osteoprotegerin (OPG), the RANK ligant (RANKL; a tumor-related necrosis factor receptor family), and osteocalcin protein immunolabels, as detected by the immunoperoxidase method and revealed with 3,3-diaminobenzidine. Drill wear and plastic deformation were analyzed by scanning electron microscopy (SEM).Results: The proteins were expressed in osteocytes of the superior bone cortical during the 40 drillings. However, in G4 and G5, a discrete increase in the expression of RANKL was observed, when compared with OPG; this increase was statistically significant in G5 (P = .016). The SEM analysis revealed major plastic deformation and drill wear in G4 and G5.Conclusion: Based on the present methodology, it may be concluded that cell viability is preserved if a less traumatic surgical protocol is used. However, the repeated use of drills alters the protein balance as of the thirtieth perforation. (C) 2008 American Association of Oral and Maxillofacial Surgeons.

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.

A dominant function of p38 mitogen-activated protein kinase signaling in receptor activator of nuclear factor-kappa B ligand expression and osteoclastogenesis induction by Aggregatibacter actinomycetemcomitans and Escherichia coli lipopolysaccharide

Background and Objective: Lipopolysaccharide from gram-negative bacteria is one of the microbial-associated molecular patterns that initiate the immune/inflammatory response, leading to the tissue destruction observed in periodontitis. The aim of this study was to evaluate the role of the p38 mitogen-activated protein kinase (MAPK) signaling pathway in lipopolysaccharide-induced receptor activator of nuclear factor-kappa B ligand (RANKL) expression by murine periodontal ligament cells.Material and Methods: Expression of RANKL and osteoprotegerin mRNA was studied by reverse transcription-polymerase chain reaction upon stimulation with lipopolysaccharide from Escherichia coli and Aggregatibacter actinomycetemcomitans. The biochemical inhibitor SB203580 was used to evaluate the contribution of the p38 MAPK signaling pathway to lipopolysaccharide-induced RANKL and osteoprotegerin expression. Stable cell lines expressing dominant-negative forms of MAPK kinase (MKK)-3 and MKK6 were generated to confirm the role of the p38 MAPK pathway. An osteoclastogenesis assay using a coculture model of the murine monocytic cell line RAW 264.7 was used to determine if osteoclast differentiation induced by lipopolysaccharide-stimulated periodontal ligament was correlated with RANKL expression.Results: Inhibiting p38 MAPK prior to lipopolysaccharide stimulation resulted in a significant decrease of RANKL mRNA expression. Osteoprotegerin mRNA expression was not affected by lipopolysaccharide or p38 MAPK. Lipopolysaccharide-stimulated periodontal ligament cells increased osteoclast differentiation, an effect that was completely blocked by osteoprotegerin and significantly decreased by inhibition of MKK3 and MKK6, upstream activators of p38 MAPK. Conditioned medium from murine periodontal ligament cultures did not increase osteoclast differentiation, indicating that periodontal ligament cells produced membrane-bound RANKL.Conclusion: Lipopolysaccharide resulted in a significant increase of RANKL in periodontal ligament cells. The p38 MAPK pathway is required for lipopolysaccharide-induced membrane-bound RANKL expression in these cells.

Role of protease-activated receptor-2 in inflammation, and its possible implications as a putative mediator of periodontitis

Proteinase-activated receptor-2 (PAR2) belongs to a novel subfamily of G-protein-coupled receptors with seven-transmembrane domains. This receptor is widely distributed throughout the body and seems to be importantly involved in inflammatory processes. PAR2 can be activated by serine proteases such as trypsin, mast cell tryptase, and bacterial proteases, such as gingipain produced by Porphyromonas gingivalis. This review describes the current stage of knowledge of the possible mechanisms that link PAR2 activation with periodontal disease, and proposes future therapeutic strategies to modulate the host response in the treatment of periodontitis.

The N terminus of the human alpha(1D)-adrenergic receptor prevents cell surface expression

We previously reported that truncation of the N-terminal 79 amino acids of alpha(1D)-adrenoceptors (Delta(1-79)alpha(1D)-ARs) greatly increases binding site density. In this study, we determined whether this effect was associated with changes in alpha(1D)-AR subcellular localization. Confocal imaging of green fluorescent protein (GFP)-tagged receptors and sucrose density gradient fractionation suggested that full-length alpha(1D)-ARs were found primarily in intracellular compartments, whereas Delta(1-79)alpha(1D)-ARs were translocated to the plasma membrane. This resulted in a 3- to 4-fold increase in intrinsic activity for stimulation of inositol phosphate formation by norepinephrine. We determined whether this effect was transplantable by creating N-terminal chimeras of alpha(1)-ARs containing the body of one subtype and the N terminus of another (alpha(1A) NT-D, alpha(1B) NT-D, alpha(1D) NT-A, and alpha(1D)NT-B). When expressed in human embryonic kidney 293 cells, radioligand binding revealed that binding densities of alpha(1A)- or alpha(1B)-ARs containing the alpha(1D)-N terminus decreased by 86 to 93%, whereas substitution of alpha(1A)- or alpha(1B)-N termini increased alpha(1D)-AR binding site density by 2- to 3-fold. Confocal microscopy showed that GFP-tagged alpha(1D)NT-B-ARs were found only on the cell surface, whereas GFP-tagged alpha(1B)NT-D-ARs were completely intracellular. Radioligand binding and confocal imaging of GFP-tagged alpha(1D)- and Delta(1-79)alpha(1D)-ARs expressed in rat aortic smooth muscle cells produced similar results, suggesting these effects are generalizable to cell types that endogenously express alpha(1D)-ARs. These findings demonstrate that the N-terminal region of alpha(1D)-ARs contain a transplantable signal that is critical for regulating formation of functional bindings, through regulating cellular localization.

COX-2 inhibition decreases VEGF expression and alveolar bone loss during the progression of experimental periodontitis in rats

Background: Vascular endothelial growth factor (VEGF) is a macromolecule of importance in inflammation that has been implicated in periodontitis. The aims of this study were to investigate VEGF expression during the progression of periodontal disease and to evaluate the effect of a preferential cyclooxygenase (COX)-2 inhibitor meloxicam on VEGF expression and alveolar bone loss in experimentally induced periodontitis.Methods: A total of 120 Wistar rats were randomly separated into groups 1 (control) and 2 (meloxicam, 3 mg/kg/day, intraperitoneally, for 3, 7, 14, or 30 days). Silk ligatures were placed at the gingival margin level of the lower right first molar of all rats. VEGF expression was assessed by reverse transcription-polymerase chain reaction (RT-PCR), Western blot (WB), and immunohistochemical (IHC) analyses. The hemiarcades were processed for histopathologic analysis. RT-PCR and WB results were submitted to analysis of variance, the Tukey test, and Pearson correlation analysis (P<0.05).Results: A reduction in alveolar bone resorption was observed in the meloxicam-treated group compared to the control group at all periods studied. There was a positive correlation between COX-2 mRNA and VEGF mRNA in the gingival tissues and periodontal disease (R = 0.80; P = 0.026). Meloxicam significantly reduced the increased mRNA VEGF expression in diseased tissues after 14 days of treatment (P = 0.023). Some alterations in VEGF receptor I mRNA expression were observed, but these were not statistically significant. VEGF protein expression in WB experiments was significantly higher in diseased sites compared to healthy sites (P<0.05). After 14 days of treatment with meloxicam, an important decrease in VEGF protein expression was detected in diseased tissues (P = 0.08). Qualitative IHC analysis revealed that VEGF protein expression was higher in diseased tissues and decreased in tissues from rats treated with meloxicam.Conclusions: The present data suggest an important role for VEGF in the progression of periodontal disease. Systemic therapy with meloxicam can modify the progression of experimentally induced periodontitis in rats by reducing VEGF expression and alveolar bone loss.

Glucose-induced insulin secretion is impaired and insulin-induced phosphorylation of the insulin receptor and insulin receptor substrate-1 are increased in protein-deficient rats

Malnutrition is related to diabetes in tropical countries. In experimental animals, protein deficiency may affect insulin secretion. However, the effect of malnutrition on insulin receptor phosphorylation and further intracellular signaling events is not known. Therefore, we decided to evaluate the rate of insulin secretion and the early molecular steps of insulin action in insulin-sensitive tissues of an animal model of protein deficiency. Pancreatic islets isolated from rats fed a standard (17%) or a low (6%) protein diet were studied for their secretory response to increasing concentrations of glucose in the culture medium. Basal as well as maximal rates of insulin secretion were significantly lower in the islets isolated from rats fed a low protein diet. Moreover, the dose-response curve to glucose was significantly shifted to the right in the islets from malnourished rats compared with islets from control rats. During an oral glucose tolerance test, there were significantly lower circulating concentrations of insulin in the serum of rats fed a low protein diet in spite of no difference in serum glucose concentration between the groups, suggesting an increased peripheral insulin sensitivity. Immunoblotting and immunoprecipitation were used to study the phosphorylation of the insulin receptor and the insulin receptor substrate-1 as well as the insulin receptor substrate-1-p85 subunit of phosphatidylinositol 3-kinase association in response to insulin. Values were greater in hind-limb muscle from rats fed a low protein diet compared with controls. No differences were detected in the total amount of protein corresponding to the insulin receptor or insulin receptor substrate-1 between muscle from rats fed the two diets. Therefore, we conclude that a decreased glucose-induced insulin secretion in pancreatic islets from protein-malnourished rats is responsible, at least in part, for an increased phosphorylation of the insulin receptor, insulin receptor substrate-1 and its association with phosphatidylinositol 3-kinase. These might represent some of the factors influencing the equilibrium in glucose concentrations observed in animal models of malnutrition and undernourished subjects.

Proteinase-activated receptor-2 (PAR2) agonist causes periodontitis in rats

Proteinase-activated receptor-2 (PAR2) is a G-protein-coupled receptor that mediates cellular responses to extracellular proteinases. Since PAR2 is expressed by oral epithelial cells, osteoblasts, and gingival fibroblasts, where its activation releases interleukin-8, we hypothesized that PAR2 activation may participate in periodontal disease in vivo. We investigated the role of PAR2 activation in periodontal disease in rats. Radiographic and enzymatic (myeloperoxidase) analysis revealed that topical application of PAR2 agonist causes periodontitis but also exacerbates existing periodontitis, leading to significant alveolar bone loss and gingival granulocyte infiltration. Inhibition of matrix metalloproteinase (MMP) and cyclo-oxygenase (COX) decreased PAR2 agonist-induced periodontitis. More specifically, the overexpression of COX-1, COX-2, MMP-2, and MMP-9 in gingival tissues suggests that they are involved in PAR 2-induced periodontitis. In conclusion, PAR2 agonist causes periodontitis in rats through a mechanism involving prostaglandin release and MMP activation. Inhibition of PAR2 may represent a novel approach to modulate host response in periodontitis.