TNF-alpha mediated transport of NF-kappaB to the nucleus is independent of the cytoskeleton-based transport system in non-neuronal cells

In unstimulated cells, proteins of the nuclear factor kappaB (NF-kappaB) transcription factor family are sequestered in the cytoplasm through interactions with IkappaB inhibitor proteins. Tumor necrosis factor alpha (TNF-alpha) activates the degradation of IkappaB-alpha and the nuclear import of cytoplasmic NF-kappaB. Nuclear localization of numerous cellular proteins is mediated by the ability of the cytoskeleton, usually microtubules, to direct their perinuclear accumulation. In a former study we have shown that activated NF-kappaB rapidly moves from distal processes in neurons towards the nucleus. The fast transport rate suggests the involvement of motor proteins in the transport of NF-kappaB. Here we address the question how NF-kappaB arrives at the nuclear membrane before import in non-neuronal cells, i.e., by diffusion alone or with the help of active transport mechanisms. Using confocal microscopy imaging and analysis of nuclear protein extracts, we show that NF-kappaB movement through the cytoplasm to the nucleus is independent of the cytoskeleton, in the three cell lines investigated here. Additionally we demonstrate that NF-kappaB p65 is not associated with the dynein/dynactin molecular motor complex. We propose that cells utilize two distinct mechanisms of NF-kappaB transport: (1) signaling via diffusion over short distances in non-neuronal cells and (2) transport via motor proteins that move along the cytoskeleton in neuronal processes where the distances between sites of NF-kappaB activation and nucleus can be vast.

Tumor necrosis factor alpha triggers proliferation of adult neural stem cells via IKK/NF-kappaB signaling

BACKGROUND: Brain inflammation has been recognized as a complex phenomenon with numerous related aspects. In addition to the very well-described neurodegenerative effect of inflammation, several studies suggest that inflammatory signals exert a potentially positive influence on neural stem cell proliferation, migration and differentiation. Tumor necrosis factor alpha (TNF-alpha) is one of the best-characterized mediators of inflammation. To date, conclusions about the action of TNF on neural stem or progenitor cells (NSCs, NPCs) have been conflicting. TNF seems to activate NSC proliferation and to inhibit their differentiation into NPCs. The purpose of the present study was to analyze the molecular signal transduction mechanisms induced by TNF and resulting in NSC proliferation. RESULTS: Here we describe for the first time the TNF-mediated signal transduction cascade in neural stem cells (NSCs) that results in increased proliferation. Moreover, we demonstrate IKK-alpha/beta-dependent proliferation and markedly up-regulated cyclin D1 expression after TNF treatment. The significant increase in proliferation in TNF-treated cells was indicated by increased neurosphere volume, increased bromodeoxyuridin (BrdU) incorporation and a higher total cell number. Furthermore, TNF strongly activated nuclear factor-kappa B (NF-kappaB) as measured by reporter gene assays and by an activity-specific antibody. Proliferation of control and TNF-treated NSCs was strongly inhibited by expression of the NF-kappaB super-repressor IkappaB-AA1. Pharmacological blockade of IkappaB ubiquitin ligase activity led to comparable decreases in NF-kappaB activity and proliferation. In addition, IKK-beta gene product knock-down via siRNA led to diminished NF-kappaB activity, attenuated cyclin D1 expression and finally decreased proliferation. In contrast, TGFbeta-activated kinase 1 (TAK-1) is partially dispensable for TNF-mediated and endogenous proliferation. Understanding stem cell proliferation is crucial for future regenerative and anti-tumor medicine. CONCLUSION: TNF-mediated activation of IKK-beta resulted in activation of NF-kappaB and was followed by up-regulation of the bona-fide target gene cyclin D1. Activation of the canonical NF-kappaB pathway resulted in strongly increased proliferation of NSCs.

Differential epigenetic reprogramming in response to specific endocrine therapies promotes cholesterol biosynthesis and cellular invasion

Endocrine therapies target the activation of the oestrogen receptor alpha (ERα) via distinct mechanisms, but it is not clear whether breast cancer cells can adapt to treatment using drug-specific mechanisms. Here we demonstrate that resistance emerges via drug-specific epigenetic reprogramming. Resistant cells display a spectrum of phenotypical changes with invasive phenotypes evolving in lines resistant to the aromatase inhibitor (AI). Orthogonal genomics analysis of reprogrammed regulatory regions identifies individual drug-induced epigenetic states involving large topologically associating domains (TADs) and the activation of super-enhancers. AI-resistant cells activate endogenous cholesterol biosynthesis (CB) through stable epigenetic activation in vitro and in vivo. Mechanistically, CB sparks the constitutive activation of oestrogen receptors alpha (ERα) in AI-resistant cells, partly via the biosynthesis of 27-hydroxycholesterol. By targeting CB using statins, ERα binding is reduced and cell invasion is prevented. Epigenomic-led stratification can predict resistance to AI in a subset of ERα-positive patients

Activation of p21-activated protein kinase alpha (alpha PAK) by hyperosmotic shock in neonatal ventricular myocytes.

The p21-activated protein kinases (PAKs) may participate in signalling from Cdc42/Rac1 to the stress-regulated MAPKs (SAPKs/JNKs and p38-/HOG-1-related-MAPKs). We characterized the expression and regulation of alpha PAK in cultured ventricular myocytes. alpha PAK was specifically immunoprecipitated from myocyte extracts. High basal alpha PAK activity was detected in unstimulated myocytes. Its activity was increased rapidly (<30 s) by hyperosmotic shock in the presence of okadaic acid, and was maximal by 3 min (187 +/- 7% relative to unstimulated cells). Endothelin-1 and interleukin-1beta, which also activate SAPKs/JNKs, did not increase alpha PAK activity and presumably act through different PAK isoforms or other mechanisms.

Characterisation of chicken riboflavin carrier protein gene structure and promoter regulation by estrogen

Estrogen is an important steroid hormone that mediates most of its effects on regulation of gene expression by binding to intracellular receptors. The consensus estrogen response element (ERE) is a 13 bp palindromic inverted repeat with a three nucleotide spacer. However, several reports suggest that many estrogen target genes are regulated by diverse elements, such as imperfect EREs and ERE half sites (ERE 1/2),which are either the proximal or the distal half of the palindrome. To gain more insight into ERE half site-mediated gene regulation, we used a region from the estrogen-regulated chicken riboflavin carrier protein (RCP) gene promoter that contains ERE half sites. Using moxestrol, an analogue of estrogen and transient transfection of deletion and mutation containing RCP promoter/reporter constructs in chicken hepatoma (LMH2A) cells, we identified an estrogen response unit (ERU) composed of two consensus ERE 1/2 sites and one non-consensus ERE 1/2 site. Mutation of any of these sites within this ERU abolishes moxestrol response. Further, the ERU is able to confer moxestrol responsiveness to a heterologous promoter. Interestingly, RCP promoter is regulated by moxestrol in estrogen responsive human MCF-7 cells, but not in other cell lines such as NIH3T3 and HepG2 despite estrogen receptor-alpha (ER-�) co transfection. Electrophoretic mobility shift assays (EMSAs) with promoter regions encompassing the half sites and nuclear extracts from LMH2A cells show the presence of a moxestrol-induced complex that is abolished by a polyclonal anti-ER� antibody. Surprisingly, estrogen receptor cannot bind to these promoter elements in isolation. Thus, there appears to be a definite requirement for some other factor(s) in addition to estrogen receptor, for the generation of a suitable response of this promoter to estrogen. Our studies therefore suggest a novel mechanism of gene regulation by estrogen, involving ERE half sites without direct binding of ER to the cognate elements.

Retinoid-Related Receptor (ROR) alpha mRNA Expression Is Altered in the Brain of Male Mice Lacking All Ligand-Binding Thyroid Hormone Receptor (TR) Isoforms

In the vertebrate brain, the thalamus serves as a relay and integration station for diverse neuronal information en route from the periphery to the cortex. Deficiency of TH during development results in severe cerebral abnormalities similar to those seen in the mouse when the retinoic acid receptor (ROR)α gene is disrupted. To investigate the effect of the thyroid hormone recep-tors (TRs) on RORalpha gene expression, we used intact male mice, in which the genes encoding the α and beta TRs have been deleted. In situ hybridization for RORalpha mRNA revealed that this gene is expressed in specific areas of the brain including the thalamus, pons, cerebellum, cortex, and hippocampus. Our quantitative data showed differences in RORalpha mRNA expression in different subthalamic nuclei between wild-type and knock-out mice. For example, the centromedial nucleus of the thalamus, which plays a role in mediating nociceptive and visceral information from the brainstem to the basal ganglia and cortical regions, has less expression of RORalpha mRNA in the knockout mice (-37%) compared to the wild-type controls. Also, in the dorsal geniculate (+72%) and lateral posterior nuclei (+58%) we found more RORalpha mRNA in dKO as compared to dWT animals. Such differences in RORalpha mRNA expression may play a role in the behavioral alterations resulting from congenital hypothyroidism.

Estrogen regulation of chicken riboflavin carrier protein gene is mediated by ERE half sites without direct binding of estrogen receptor

Estrogen is an important steroid hormone that mediates most of its effects on regulation of gene expression by binding to intracellular receptors. The consensus estrogen response element (ERE) is a 13 bp palindromic inverted repeat with a three nucleotide spacer. However, several reports suggest that many estrogen target genes are regulated by diverse elements, such as imperfect EREs and ERE half sites (ERE 1/2), which are either the proximal or the distal half of the palindrome. To gain more insight into ERE half site-mediated gene regulation, we used a region from the estrogen-regulated chicken riboflavin carrier protein (RCP) gene promoter that contains ERE half sites. Using moxestrol, an analogue of estrogen and transient transfection of deletion and mutation containing RCP promoter/reporter constructs in chicken hepatoma (LMH2A) cells, we identified an estrogen response unit (ERU) composed of two consensus ERE 1/2 sites and one non-consensus ERE 1/2 site. Mutation of any of these sites within this ERU abolishes moxestrol response. Further, the ERU is able to confer moxestrol responsiveness to a heterologous promoter. Interestingly, RCP promoter is regulated by moxestrol in estrogen responsive human MCF-7 cells, but not in other cell lines such as NIH3T3 and HepG2 despite estrogen receptor-alpha (ER-�) co transfection. Electrophoretic mobility shift assays (EMSAs) with promoter regions encompassing the half sites and nuclear extracts from LMH2A cells show the presence of a moxestrol-induced complex that is abolished by a polyclonal anti-ER� antibody. Surprisingly, estrogen receptor cannot bind to these promoter elements in isolation. Thus, there appears to be a definite requirement for some other factor(s) in addition to estrogen receptor, for the generation of a suitable response of this promoter to estrogen. Our studies therefore suggest a novel mechanism of gene regulation by estrogen, involving ERE half sites without direct binding of ER to the cognate elements.

Influence of pulse repetition rate of Er : YAG laser and dentin depth on tensile bond strength of dentin-resin interface

The study evaluated the in vitro influence of pulse-repetition rate of Er:YAG laser and dentin depth on tensile bond strength of dentin-resin interface. Dentin surfaces of buccal or lingual surfaces from human third molars were submitted to tensile test in different depths (superficial, 1.0 and 1.5 mm) of the same dental area, using the same sample. Surface treatments were acid conditioning solely (control) and Er:YAG laser irradiation (80 mJ) followed by acid conditioning, with different pulse-repetition rates (1, 2, 3, or 4 Hz). Single bond/Z-250 system was used. The samples were stored in distilled water at 37 degrees C for 24 h, and then the first test (superficial dentine) was performed. The bond failures were analyzed. Following, the specimens were identified, grounded until 1.0- and 1.5-mm depths, submitted again to the treatments and to the second and, after that, to third-bond tests on a similar procedure and failure analysis. ANOVA and Tukey test demonstrated a significant difference (p < 0.001) for treatment and treatment X depth interaction (p < 0.05). The tested depths did not show influence (p > 0.05) on the bond strength of dentin-resin interface. It may be concluded that Er:YAG laser with 1, 2, 3, or 4 Hz combined with acid conditioning did not increase the resin tensile bond strength to dentin, regardless of dentin depth. (C) 2007 Wiley Periodicals, Inc.

Stromal-derived factor-1 alpha (CXCL12) levels increase in periodontal disease

Background: The CXC chemokine receptor 4 (CXCR4) and its ligand, stromal cell-derived factor-1 (SDF-1 alpha or CXC chemokine ligand 12) are involved in the trafficking of leukocytes into and out of extravascular tissues. The purpose of this study was to determine whether SDF-1 alpha secreted by host cells plays a role in recruiting inflammatory cells into the periodontia during local inflammation. Methods: SDF-1 alpha levels were determined by enzyme-linked immunosorbent assay in gingival crevicular fluid (GCF) of 24 individuals with periodontitis versus healthy individuals in tissue biopsies and in a preclinical rat model of Porphyromonas gingivalis lipopolysaccharide-induced experimental bone loss. Neutrophil chemotaxis assays were also used to evaluate whether SDF-1 alpha plays a role in the recruitment of host cells at periodontal lesions. Results: Subjects with periodontal disease had higher levels of SDF-1 alpha in their GCF compared to healthy subjects. Subjects with periodontal disease who underwent mechanical therapy demonstrated decreased levels of SDF-1 alpha. Immunohistologic staining showed that SDF-1 alpha and CXCR4 levels were elevated in samples obtained from periodontally compromised individuals. Similar results were observed in the rodent model. Neutrophil migration was enhanced in the presence of SDF-1 alpha, mimicking immune cell migration in periodontal lesions. Conclusions: SDF-1 alpha may be involved in the immune defense pathway activated during periodontal disease. Upon the development of diseased tissues, SDF-1 alpha levels increase and may recruit host defensive cells into sites of inflammation. These studies suggest that SDF-1 alpha may be a useful biomarker for the identification of periodontal disease progression.

Nitric oxide detection in cell culture exposed to LPS after Er:YAG laser irradiation

P>Aim To evaluate in vitro the effect of calcium hydroxide [Ca(OH)(2)] and Er:YAG laser on bacterial endotoxin [also known as lipopolysaccharide (LPS)] as determined by nitric oxide (NO) detection in J774 murine macrophage cell line culture. Methodology Samples of LPS solution (50 mu gmL-1), Ca(OH)(2) suspension (25 mg mL-1) and LPS suspension with Ca(OH)(2) were prepared. The studied groups were: I - LPS (control); II - LPS + Ca(OH)(2); III - LPS + Er:YAG laser (15 Hz 140 mJ); IV - LPS + Er:YAG laser (15 Hz 200 mJ); V - LPS + Er:YAG laser (15 Hz 250 mJ), VI - Pyrogen-free water; VII - Ca(OH)(2). Murine macrophage J774 cells were plated and 10 mu L of the samples were added to each well. The supernatants were collected for NO detection by the Griess reaction. Data were analysed statistically by one-way anova and Tukey`s test at 5% significance level. Results The mean and SE (in mu mol L-1) values of NO release were: I - 10.48 +/- 0.58, II - 6.41 +/- 0.90, III - 10.2 +/- 0.60, IV - 8.35 +/- 0.40, V - 10.40 +/- 0.53, VI - 3.75 +/- 0.70, VII - 6.44 +/- 0.60; and the values for the same experiment repeated after 1 week were: I - 21.20 +/- 1.50, II - 9.10 +/- 0.60, III - 19.50 +/- 1.00, IV - 18.50 +/- 0.60, V - 21.30 +/- 0.90, VI - 2.00 +/- 0.20, VII - 6.80 +/- 1.70. There was no significant difference (P > 0.05) between the control and the laser-treated groups (III, IV and V), or comparing groups II, VI and VII to each other (P > 0.05). Group I had significantly higher NO release than group II (P < 0.05). Groups II and VI had similar NO release (P > 0.05). Conclusions Calcium hydroxide inactivated the bacterial endotoxin (LPS) whereas none of the Er:YAG laser parameter settings had the same effectiveness.

TNF-alpha Promotes an Odontoblastic Phenotype in Dental Pulp Cells

Dental pulp cells can differentiate toward an odontoblastic phenotype to produce reparative dentin beneath caries lesions. However, the mechanisms involved in pulp cell differentiation under pro-inflammatory stimuli have not been well-explored. Thus, we hypothesized that the pro-inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) could be a mediator involved in dental pulp cell differentiation toward an odontoblastic phenotype. We observed that TNF-alpha-challenged pulp cells exhibited increased mineralization and early and increased expression of dentin phosphoprotein (DPP), dentin sialoprotein (DSP), dentin matrix protein-1, and osteocalcin during a phase of reduced matrix metalloproteinase (MMP) expression. We investigated whether these events were related and found that p38, a mitogen-activated protein kinase, differentially regulated MMP-1 and DSP/DPP expression and mediated mineralization upon TNF-alpha treatment. These findings indicate that TNF-alpha stimulates differentiation of dental pulp cells toward an odontoblastic phenotype via p38, while negatively regulating MMP-1 expression.

SEM analysis of enamel surface treated by Er : YAG laser: Influence of irradiation distance

Background: Depending on the distance of laser tip to dental surface a specific morphological pattern should be expected. However, there have been limited reports that correlate the Er:YAG irradiation distance with dental morphology. Purpose: To assess the influence of Er:YAG laser irradiation distance on enamel morphology, by means of scanning electron microscopy (SEM). Methods: Sixty human third molars were employed to obtain discs (congruent to 1 mm thick) that were randomly assigned to six groups (n = 10). Five groups received Er:YAG laser irradiation (80 mJ/2 Hz) for 20 s, according to the irradiation distance: 11, 12, 14, 16, or 17 mm. and the control group was treated with 37% phosphoric acid for 15 s. The laser-irradiated discs were bisected. One hemi-disc was separated for superficial analysis without subsequent acid etching, and the other one, received the phosphoric acid for 15 s. Samples were prepared for SEM. Results: Laser irradiation at 11 and 12 min provided an evident ablation of enamel, with evident fissures and some fused areas. At 14, 16 and 17 mm the superficial topography was flatter than in the other distances. The subsequent acid etching on the lased-surface partially removed the disorganized tissue. Conclusions: Er:YAG laser in defocused mode promoted slight morphological alterations and seems more suitable for enamel conditioning than focused irradiation. The application of phosphoric acid on lased-enamel surface, regardless of the irradiation distance, decreased the superficial irregularities.

Influence of energy and pulse repetition rate of Er : YAG laser on enamel ablation ability and morphological analysis of the laser-irradiated surface

Objective: To assess the influence of energy and pulse repetition rate of Er:YAG laser on the enamel ablation ability and substrate morphology. Methods: Fifteen crowns of molars were sectioned in four fragments, providing 60 samples, which were ground to flatten the enamel surface. The initial mass was obtained by weighing the fragments. The specimens were hydrated for I h, fixed, and a 3-mm-diameter area was delimited. Twelve groups were randomly formed according to the combination of laser energies (200, 250, 300, or 350 mJ) and pulse repetition rates (2, 3, or 4 Hz). The final mass was obtained and mass loss was calculated by the difference between the initial and final mass. The specimens were prepared for SEM. Data were submitted to ANOVA and Scheffe test. Results: The 4 Hz frequency resulted in higher mass loss and was statistically different from 2 and 3 Hz (p < 0.05). The increase of frequency produced more melted areas, cracks, and unselective and deeper ablation. The 350 mJ energy promoted greater mass loss, similar to 300 mJ. Conclusions: The pulse repetition rate influenced more intensively the mass loss and morphological alteration. Among the tested parameters, 350 mJ/3 Hz improved the ability of enamel ablation with less surface morphological alterations. (C) 2007 Wiley Periodicals, Inc. J Biomed Mater Res.