Expression of suppressor of cytokine signaling 1 and 3 in ligature-induced periodontitis in rats

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

Sequential IL-23 and IL-17 and increased Mmp8 and Mmp14 expression characterize the progression of an experimental model of periodontal disease in type 1 diabetes

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

Interleukin-1 beta and Interleukin-6 Expression and Gene Polymorphisms in Subjects with Peri-Implant Disease

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Expression of protease nexin-1 and plasminogen activators during follicular growth and the periovulatory period in cattle

Extracellular matrix remodeling occurs during ovarian follicular development, mediated by plasminogen activators (PAs) and PA inhibitors including protease nexin-1 (PN-1). In the present study we measured expression/activity of the PA system in bovine follicles at different stages of development by timed collection of ovaries during the first follicular wave and during the periovulatory period, and in follicles collected from an abattoir. The abundance of mRNA encoding PN-1, tissue-type PA (tPA), urokinase (uPA) and PA inhibitor-1 (PAI-1) were initially upregulated by human chorionic gonadotropin (hCG) in bovine preovulatory follicular wall homogenates. PN-1, PAI-1 and tPA mRNA expression then decreased near the expected time of ovulation, whereas uPA mRNA levels remained high. PN-1 concentration in follicular fluid (FF) decreased and reached the lowest level at the time of ovulation, whereas plasmin activity in FF increased significantly after hCG. Follicles collected from the abattoir were classified as non-atretic, early-atretic or atretic based on FF estradiol and progesterone content: PN-1 protein levels in FF were significantly higher in non-atretic than in atretic follicles, and plasmin activity was correspondingly higher in the atretic follicles. No changes in PN-1 levels in FF were observed during the growth of pre-deviation follicles early in a follicular wave. These results indicate that PN-1 may be involved in the process of atresia in non-ovulatory dominant follicles and the prevention of precocious proteolysis in periovulatory follicles.

Heart failure increases atrogin-1 and MuRF1 gene expression in skeletal muscle with fiber type-specific atrophy

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

Time-dependent expression of annexin 1 in a model of chronic granulomatous inflammation

Objective and design: To determine the expression pattern and distribution of the glucocorticoid-inducible protein annexin 1 (ANXA1) in a murine model of chronic granulomatous inflammation.Materials or subjects: TO Mouse.Treatment: Chronic granulomatous inflammation was induced by injecting into dorsal sub-cutaneous air-pouches in mice, a mixture of croton oil and Freund's complete adjuvant (CO/FCA).Methods: Western and northern analysis, corticosterone assay, and immunohistochemistry. Statistical analysis was performed using ANOVA followed by Tukey's pair-wise comparisons or Dunnett's multiple comparisons.Results: ANXA1 protein levels changed significantly throughout the 4-week time course, with an initial peak at day 7 and a later elevation at 28 days. ANXA1 mRNA levels peaked at days 1 and 3, with a significant decline at day 7 followed by an upward trend to day 28. Plasma corticosterone measurements taken throughout the time course revealed an increase from 14 days onward, suggesting that corticosterone does not influence ANXA1 expression during the initial stages of the model. Immunogold staining revealed that ANXA1 expression in the inflamed tissue was mainly in extravasated neutrophils, with intact protein (37 kDa) being predominantly observed on the cell membrane.Conclusions: the pattern of ANXA1 expression indicates that infiltrated neutrophils are responsible for the majority of ANXA1 present both at early and later stages of this model of granulomatous inflammation.

Annexin 1: differential expression in tumor and mast cells in human larynx cancer

Annexin 1 protein (ANXA1) expression was evaluated in tumor and mast cells in human larynx cancer and control epithelium. The effect of the exogenous ANXA1 (peptide Ac 2-26) was also examined during the cellular growth of the Hep-2 human larynx epidermoid carcinoma cell line. This peptide inhibited the proliferation of the Hep-2 cells within 144 hr. In surgical tissue specimens from 20 patients with larynx cancer, ultrastructural immunocytochemistry analysis showed in vivo down-regulation of ANXA1 expression in the tumor and increased in mast cells and Hep-2 cells treated with peptide Ac2-26. Combined in vivo and in vitro analysis demonstrated that ANXA1 plays a regulatory role in laryngeal cancer cell growth. We believe that a better understanding of the regulatory mechanisms of ANXA1 in tumor and mast cells may lead to future biological targets for the therapeutic intervention of human larynx cancer. (c) 2007 Wiley-Liss, Inc.

Interaction of human neutrophils with endothelial cells regulates the expression of endogenous proteins annexin 1, galectin-1 and galectin-3

Annexin 1 (ANXA1), galectin-1 (Gal-1) and galectin-3 (Gal-3) proteins have been identified as important mediators that promote or inhibit leukocyte migration. The expression of these proteins was studied in human neutrophils and endothelial cells (ECs) during a transmigration process induced by IL-8. Upon neutrophil adhesion to EC, a significant increase in the cleaved ANXA1 (LCS3, raised against all ANXA1 isoforms) expression was detected in the plasma membrane of adhered neutrophils and ECs compared to intact ANXA1 isoform (LCPS1, against N-terminus of protein). Adherent neutrophils had elevated Gal-3 levels in the nucleus and cytoplasm, and ECs in their plasma membranes. In contrast, a decrease in the total amounts of Gal-1 was detected in migrated compared to non-migrated neutrophils. Therefore, ANXA1 and Gal-3 changed in their content and localization when neutrophils adhere to endothelia, suggesting a process of sensitive-balance between two endogenous anti- and pro-inflammatory mediators. (c) 2006 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved.

Expression pattern of claudins 1 and 3-an auxiliary tool in predicting behavior of mucoepidermoid carcinoma of salivary gland origin

Claudins (CLDNs) are a family of membrane proteins important for permeability of tight junctions. They have also been implicated in carcinogenesis and tumor progression. We analyzed patterns of distribution and intensity of expression of CLDNs 1, 3, 4, and 7 in mucoepidermoid carcinoma (MEC) of salivary gland in 39 patients. Correlations between the expression of CLDNs, tumor grade, and survival were explored. In immunohistochemical analysis, high expression of CLDN 1 was seen in low-grade MEC, and it appeared to be a suitable auxiliary marker of good prognosis. It classified MEC similarly to histological grading in 89.7% of cases (p=0.001). High CLDN 3 expression was seen in intermediate-and high-grade MEC, while it was low in low-grade MEC. CLDN 3 intensity correctly categorized tumors into grades in 71.8% of cases (p=0.017). However, in multivariate analysis CLDN 1 and CLDN 3 did not achieve significance over tumor grade in predicting patient behavior. We conclude that analysis of staining intensities of CLDN 1 and 3 is useful as an auxiliary diagnostic and prognostic tool in patients with salivary gland MEC.

Abnormal expression of bcl-2 and bax in rat tongue mucosa during the development of squamous cell carcinoma induced by 4-nitroquinoline 1-oxide

4-Nitroquinoline 1-oxide (4NQO)-induced rat tongue carcinogenesis is a useful model for studying oral squamous cell carcinoma. The aim of this study was to investigate the expression of bcl-2 and bax during tongue carcinogenesis induced by 4NQO. Male Wistar rats were distributed into three groups of 10 animals each and treated with 50 ppm 4NQO solution through their drinking water for 4, 12 or 20 weeks. Ten animals were used as negative control. Although no histological changes were induced in the epithelium after 4 weeks of carcinogen exposure, bcl-2 and bax were over-expressed (P < 0.01) in all layers of the 'normal' epithelium. The expression levels were the same in all layers of epithelium for both the antibodies used (bcl-2 or bax). In dysplastic lesions at 12 weeks following carcinogen administration, the levels of bcl-2 and bax expression did not increase when compared to negative control with the immunoreactivity for bcl-2 being restricted to the superficial layer of epithelium. In well-differentiated squamous cell carcinoma induced after 20 weeks of treatment with 4NQO, bcl-2 was expressed in some cells of tumour islands. on the other hand, immunostaining for bax was widely observed at the tumour nests. The labelling index for bcl-2 and bax showed an increase (P < 0.05) after only 4 weeks of 4NQO administration. In conclusion, our results suggest that abnormalities in the apoptosis pathways are associated with the development of persistent clones of mutated-epithelial cells in the oral mucosa. Bcl-2 and bax expression appears to be associated with a risk factor in the progression of oral cancer.

MKK3/6-p38 MAPK negatively regulates murine MMP-13 gene expression induced by IL-1 beta and TNF-alpha in immortalized periodontal ligament fibroblasts

Matrix metalloprotease-13 (MMP-13) or collagenase-3 is involved in a number of pathologic processes such as tumor metastasis and angiogenesis, osteoarthritis, rheumatoid arthritis and periodontal diseases. These conditions are associated with extensive degradation of both connective tissue and bone. This report examines gene regulation mechanisms and signal transduction pathways involved in Mmp-13 expression induced by proinflammatory cytokines in periodontal ligament (PDL) fibroblasts. Mmp-13 mRNA expression was increased 10.7 and 9.5 fold after stimulation with IL-1 beta (5 ng/mL) and TNF-alpha (10 ng/mL), respectively. However, inhibition of p38 MAPKinase with SB203580 resulted in significant (p < 0.001) induction (23.2 and 18.1 fold, respectively) of Mmp-13 mRNA as assessed by real time PCR. Negative regulation of IL-1 induced Mmp-13 expression was confirmed by inhibiting p38 MAPK gene expression with siRNA. Transient transfection of dominant negative forms of MKK3 and MKK6 also resulted in increased levels of Mmp-13 mRNA after IL-1 beta stimulation. Mmp-13 mRNA expression induced by TNF-alpha was decreased by JNK and ERK inhibition. Western blot and zymogram analysis indicated that Mmp-13 protein expression induced by the proinflammatory cytokines were also upregulated by inhibition of p38 MAPK. Reporter gene experiments using stable cell lines harboring 660-bp sequence of the murine Mmp-13 proximal promoter indicated that transcriptional mechanisms were at least partially involved in this negative regulation of Mmp-13 expression by p38 MAPK and upstream MKK3/6. These results suggest a negative transcriptional regulatory mechanism mediated by p38 MAPK and upstream MKK3/6 on Mmp-13 expression induced by proinflammatory cytokines in PDL fibroblasts. (c) 2005 Elsevier B.V./International Society of Matrix Biology. All rights reserved.

Myenteric Denervation Downregulates Galectin-1 and-3 Expression in Gastric Carcinogenesis

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

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.