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.

Critical protective role for annexin 1 gene expression in the endotoxemic murine microcirculation

The inflammatory response is a protective process of the body to counteract xenobiotic penetration and injury, although in disease this response can become deregulated. There are endogenous biochemical pathways that operate in the host to keep inflammation under control. Here we demonstrate that the counter-regulator annexin 1 (AnxA1) is critical for controlling experimental endotoxemia. Lipopolysaccharide (LPS) markedly activated the AnxA1 gene in epithelial cells, neutrophils, and peritoneal, mesenteric, and alveolar macrophages-cell types known to function in experimental endotoxemia. Administration of LPS to AnxA1-deficient mice produced a toxic response characterized by organ injury and lethality within 48 hours, a phenotype rescued by exogenous application of low doses of the protein. In the absence of AnxA1, LPS generated a deregulated cellular and cytokine response with a marked degree of leukocyte adhesion in the microcirculation. Analysis of LPS receptor expression in AnxA1-null macrophages indicated an aberrant expression of Toll-like receptor 4. In conclusion, this study has detailed cellular and biochemical alterations associated with AnxA1 gene deletion and highlighted the impact of this protective circuit for the correct functioning of the homeostatic response to sublethal doses of LPS. Copyright © American Society for Investigative Pathology.

Role of annexin 1 gene expression in mouse craniofacial bone development

BACKGROUND: Annexin 1 is a 37-kDa protein that has complex intra- and extracellular effects. To discover whether the absence of this protein alters bone development, we monitored this event in the annexin-A1 null mice in comparison with littermate wild-type controls. METHODS: Radiographic and densitometry methods were used for the assessment of bone in annexin-A1 null mice at a gross level. We used whole-skeleton staining, histological analysis, and Western blotting techniques to monitor changes at the tissue and cellular levels. RESULTS: There were no gross differences in the appendicular skeleton between the genotypes, but an anomalous development of the skull was observed in the annexin-A1 null mice. This was characterized in the newborn annexin-A1 null animals by a delayed intramembranous ossification of the skull, incomplete fusion of the interfrontal suture and palatine bone, and the presence of an abnormal suture structure. The annexin-A1 gene was shown to be active in osteocytes during this phase and COX-2 was abundantly expressed in cartilage and bone taken from annexin-A1 null mice. CONCLUSIONS: Expression of the annexin-A1 gene is important for the normal development of the skull in mice, possibly through the regulation of osteoblast differentiation and a secondary effect on the expression of components of the cPLA2-COX-2 system. © 2007 Wiley-Liss, Inc.

Morphologic changes and the expression of alpha-melanocyte stimulating hormone and melanocortin-1 receptor in melasma lesions: A comparative study

Melasma is a common acquired symmetrical hypermelanosis characterized by irregular light- to dark-brown macules on sun-exposed skin areas. The literature shows few studies on its physiopathogeny. However, changes in α-melanocyte stimulating hormone (α-MSH) secretion and melanocortin-1 receptor (MC1-R) expression may play a role to trigger this condition. Biopsies were taken from both melasma skin and adjacent perilesional normal skin of 44 patients. The biopsies were submitted for hematoxylin and eosin and Fontana-Masson staining and immunohistochemistry with Melan-A, α-MSH, and MC1-R, and processed for transmission electron microscopy. In some cases, they were submitted to MC1-R gene expression analysis by real-time polymerase chain reaction. Increased lymphohistiocytic infiltrate and solar elastosis, higher epidermal melanin were observed in melasma skin. Electron microscopy revealed a greater number of mature melanosomes in keratinocytes and melanocytes, and more prominent cytoplasmic organelles in melasma skin. There was no difference in melanocyte number between areas. However, melanocytes were larger and more dendritic in melasma skin. Immunohistochemistry with α-MSH and MC1-R showed significant labeling in melasmic epidermis but MC1-R messenger ribonucleic acid (RNAm) did not show significant quantitative difference between melasma and normal skin. © 2010 by Lippincott Williams & Wilkins.

The expression of aquaporins 1 and 9 in adult rat epididymis is perturbed by chronic exposure to ethanol

Aquaporins (AQPs), notably AQP-1 and AQP-9, may contribute to reabsorption of fluid and solute across the epididymis. Ethanol is related to be a toxicant affecting directly or indirectly the epididymis and the sperm motility. This study examined the expression of AQP-1 and AQP-9 in adult epididymis of the UChA and UChB 10% (v/v) ethanol-preferring rats, focusing the ethanol-induced hormonal disturbances upon the regulation of these AQPs. Chronic ethanol intake significantly decreased body weight, while UChA and UChB rats displayed a marked loss of epididymal weights. Both ethanol-consuming animals had a severe reduction of testosterone levels, whereas LH and 17β-estradiol were unchanged. Throughout the epididymis, a strong reaction to AQP-1 was observed in myoid and endothelial cells of the UChB ethanol-preferring rats, differently from a moderate intensity in the initial segment of the UChA rats. In addition, AQP-9 showed a strong immunoreaction in the apical membrane of principal cells at initial segment. In cauda epididymis, the level of AQP-9 was reduced along the microvillus projections in both UChA and UChB rats compared to controls. We conclude that chronic ethanol consumption modulates the androgen levels, thereby modifying the expression pattern of AQP-1 and 9 in the epididymis. © 2011 Elsevier Ltd.

The effect of laminin-1-doped nanoroughened implant surfaces: Gene expression and morphological evaluation

Aim. This study aimed to observe the morphological and molecular effect of laminin-1 doping to nanostructured implant surfaces in a rabbit model. Materials and Methods. Nanostructured implants were coated with laminin-1 (test; dilution, 100 g/mL) and inserted into the rabbit tibiae. Noncoated implants were used as controls. After 2 weeks of healing, the implants were removed and subjected to morphological analysis using scanning electron microscopy (SEM) and gene expression analysis using the real-time reverse transcriptase-polymerase chain reaction (RT-PCR). Results. SEM revealed bony tissue attachment for both control and test implants. Real-time RT-PCR analysis showed that the expression of osteoblast markers RUNX-2, osteocalcin, alkaline phosphatase, and collagen I was higher (1.62-fold, 1.53-fold, 1.97-fold, and 1.04-fold, resp.) for the implants modified by laminin-1 relative to the control. All osteoclast markers investigated in the study presented higher expression on the test implants than controls as follows: tartrate-resistant acid phosphatase (1.67-fold), calcitonin receptor (1.35-fold), and ATPase (1.25-fold). The test implants demonstrated higher expression of inflammatory markers interleukin-10 (1.53-fold) and tumour necrosis factor-α (1.61-fold) relative to controls. Conclusion. The protein-doped surface showed higher gene expression of typical genes involved in the osseointegration cascade than the control surface. © 2012 Humberto Osvaldo Schwartz-Filho et al.

Evaluation of the expression of p53, MDM2, and SUMO-1 in oral lichen planus

Objective: The objective of this study was to compare the expression of proteins p53, MDM2, and SUMO-1 in oral lichen planus (OLP) lesions, epithelial dysplasia, and squamous cell carcinoma. Materials and Methods: The sample consisted of the following five groups of cheek mucosa lesions: normal mucosa (NM), inflammatory fibrous hyperplasia (IFH), lichen planus, epithelial dysplasia, and squamous cell carcinoma. The tissue samples were stained with hematoxylin-eosin and submitted to immunohistochemistry using anti-p53, anti-MDM2, and anti-SUMO-1 antibodies. Results: The results of this study demonstrated similar expression of p53 and MDM2 between OLP, oral epithelial dysplasia and, to a lesser extent, between OLP and oral squamous cell carcinoma (OSCC). However, for SUMO-1 a similar expression was observed in OLP, NM, and IFH. Conclusions: The results demonstrated overexpression of important proteins (p53 and MDM2) related to regulatory mechanisms of apoptosis in OLP, suggesting that there is a favorable environment for malignant transformation. The expression of SUMO-1 in OLP was similar to NM and IFH, suggesting that alterations of this protein occur at later stages of carcinogenesis, because important overexpression occurred in oral epithelial dysplasia and OSCC. © 2013 John Wiley & Sons A/S.

Expression of annexin-A1 and galectin-1 anti-inflammatory proteins and mRNA in chronic gastritis and gastric cancer

Objective. The anti-inflammatory proteins annexin-A1 and galectin-1 have been associated with tumor progression. This scenario prompted us to investigate the relationship between the gene and protein expression of annexin-A1 (ANXA1/AnxA1) and galectin-1 (LGALS1/Gal-1) in an inflammatory gastric lesion as chronic gastritis (CG) and gastric adenocarcinoma (GA) and its association with H. pylori infection. Methods. We analyzed 40 samples of CG, 20 of GA, and 10 of normal mucosa (C) by the quantitative real-time PCR (qPCR) technique and the immunohistochemistry assay. Results. High ANXA1 mRNA expression levels were observed in 90% (36/40) of CG cases (mean relative quantification RQ = 4.26 ± 2.03) and in 80% (16/20) of GA cases (mean RQ = 4.38 ± 4.77). However, LGALS1 mRNA levels were high (mean RQ = 2.44 ± 3.26) in 60% (12/20) of the GA cases, while low expression was found in CG (mean RQ = 0.43 ± 3.13; P < 0.01). Normal mucosa showed modest immunoreactivity in stroma but not in epithelium, while stroma and epithelium displayed an intense immunostaining in CG and GA for both proteins. Conclusion. These results have provided evidence that galectin-1 and mainly annexin-A1 are overexpressed in both gastritis and gastric cancer, suggesting a strong association of these proteins with chronic gastric inflammation and carcinogenesis. © 2013 Yvana Cristina Jorge et al.

Orthodontic force increases interleukin-1β and tumor necrosis factor-α expression and alveolar bone loss in periodontitis

Background: The present study aims to evaluate the effects of orthodontic movement (OM) on the periodontal tissues of rats with ligature-induced periodontal disease. Methods: Eighty-eight rats were divided into four groups: 1) negative control (sham operated); 2) periodontal disease; 3) OM; and 4) periodontal disease followed by OM (OMP). Rats were sacrificed 3 hours or 1, 3, or 7 days after OM commencement. Bone volume fraction (BVF) and bone mineral density (BMD) were assessed in hemimaxillae by microcomputed tomography analysis. Expression of the proinflammatory cytokines interleukin (IL)-1β and tumor necrosis factor (TNF)-α were evaluated in gingival samples by quantitative polymerase chain reaction and enzyme-linked immunosorbent assay, and in the furcation region by immunohistochemistry analysis (IHC). Results: The OMP group had lower BVF and BMD levels compared to the other groups at day 7 (P <0.05). Maximum messenger ribonucleic acid expression of both cytokines was observed in the OMP group at day 1 (P <0.05). In the same period, all proteins were expressed in high levels for all test groups compared to the control group. The number of cells positive for IL-1β and TNF-α by IHC was highest in the OMP group at day 1, with progressive reduction thereafter. Conclusion: The results suggest that OM acts synergistically with periodontal disease in periodontal breakdown through upregulation of proinflammatory cytokines.

Effect of glycerol on GHR and IGF-1 gene expression in breast muscle and on growth of Japanese meat quails

We evaluated messenger RNA (mRNA) expression of the growth-hormone (GHR) and insulin-like growth factor (IGF-1) genes in 28-day-old Japanese meat quails fed diets containing 0, 8, or 12% dietary glycerol in substitution of corn. Total RNA was extracted from the breast muscle and the DNA was amplified with specific primers using real-time PCR. Feed conversion ratio and feed intake were evaluated. The birds fed 8 and 12% glycerol presented higher IGF-1 mRNA expression [0.059 and 0.049 arbitrary units (AU), respectively] relative to those not fed with glycerol (0.029 AU), while 12% glycerol reduced GHR mRNA expression (0.022 AU). Dietary inclusion of 8% glycerol promoted similar performance results (feed conversion) as the diet with no glycerol. We conclude that inclusion of glycerol in the diet affects GHR and IGF-1 gene expression in Japanese meat quails. However, considering the performance results and the expression of the GHR and IGF-1 genes, 8% glycerol may be safely included in the diet of meat quails. © FUNPEC-RP.

Gene expression and protein localization of TLR-1, -2, -4 and -6 in amniochorion membranes of pregnancies complicated by histologic chorioamnionitis

Objectives: To determine whether histologic chorioamnionitis is associated with changes in gene expression of TLR-1, -2, -4 and -6, and to describe the localization of these receptors in fetal membranes. Study design: A total of 135 amniochorion membranes with or without histologic chorioamnionitis from preterm or term deliveries were included. Fragments of membranes were submitted to total RNA extraction. RNA was reverse transcribed and the quantification of TLRs expression measured by real time PCR. Results: All amniochorion membranes expressed TLR-1 and TLR-4, whereas 99.1% of membranes expressed TLR-2 and 77.4% expressed TLR-6. TLR-1 and TLR-2 expressions were significantly higher in membranes with histologic chorioamnionitis as compared to membranes without chorioamnionitis in preterm pregnancies (p = 0.003 and p < 0.001, respectively). Among the membranes of term pregnancies there were no differences in the expressions of such receptors regardless of inflammatory status. Regarding TLR-4 and TLR-6 expression, there was no difference among membranes with or without histologic chorioamnionitis, regardless gestational age at delivery. TLR-1, TLR-2, TLR-4 and TLR-6 expressions were observed in amniotic epithelial, chorionic and decidual cells. Conclusion: Amniochorion membranes express TLR-1, TLR-2, TLR-4 and TLR-6 and increased expression of TLR-1 and TLR-2 is related to the presence of histologic chorioamnionitis in preterm pregnancies. This study provides further evidence that amniochorion membranes act as a mechanical barrier to microorganisms and as components of the innate immune system. © 2013 Elsevier B.V. All rights reserved.

Association of matrix metalloproteinase inducer (EMMPRIN) with the expression of matrix metalloproteinases-1, -2 and -9 during periapical lesion development

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