Ezrin-related phosphoinositide pathway modifies RhoA and Rac1 in human osteosarcoma cell lines

Selected Phosphoinositide-specific Phospholipase C (PI-PLC) enzymes occupy the convergence point of the broad range of pathways that promote Rho and Ras GTPase mediated signalling, which also regulate the activation of ezrin, a member of the ezrin-radixin-moesin (ERM) proteins family involved in the metastatic osteosarcoma spread. Previous studies described that in distinct human osteosarcoma cell lines ezrin networks the PI-PLC with complex interplay controlling the expression of the PLC genes, which codify for PI-PLC enzymes. In the present study, we analyzed the expression and the sub-cellular distribution of RhoA and Rac1 respectively after ezrin silencing and after PI-PLC ε silencing, in order to investigate whether ezrin-RhoGTPAses signalling might involve one or more specific PI-PLC isoforms in cultured 143B and Hs888 human osteosarcoma cell lines. In the present experiments, both ezrin and PLCE gene silencing had different effects upon RhoA and Rac1 expression and sub-cellular localization. Displacements of Ezrin and of RhoA localization were observed, probably playing functional roles

Immunohistochemical localization and mRNA expression of matrix Gla protein and fetuin-A in bone biopsies of hemodialysis patients

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Expression and localization of Phosphoinositide-specific Phospholipase C enzymes in polarized macrophages

The phenotypic and functional diversity of macrophages depends on differentiating programs being developed during the cells’ lives. Great interest was addressed to identify the signal transduction pathways acting in macrophage polarization, including the phosphoinositide (PI) system and related phospholipase C (PLC) family of enzymes. Enzymes belonging to the PLC family are strictly tissue specific and the expression panel, as well as the subcellular localization differs in quiescent cells compared to the pathological counterpart. We analyzed the expression of PLC enzymes in unpolarized (M0), M1 and M2 macrophages to list the isoforms expressed in the polarized macrophages and their subcellular localization. Our results confirmed that macrophages express a wide number of PLC isoforms. All PLC enzymes were detected within both M1 and M2 cells, but not in M0 cells. M0, as well as M1 and M2 cells own a specific panel of expression, different for both genes’ mRNA expression and intracellular localization of PLC enzymes. PLC enzymes might play a complex role in macrophages during inflammation and probably also during polarization

Involvement of substance P (SP) and its related NK1 receptor in primary Sjögren’s syndrome (pSS) pathogenesis

Primary Sjogren's Syndrome (pSS) is a systemic autoimmune disease that primarily attacks the lacrimal and salivary glands, resulting in impaired secretory function characterized by xerostomia and xerophthalmia. Patients with pSS have been shown to have impaired salivary gland innervation and altered circulating levels of neuropeptides thought to be a cause of decreased salivation, including substance P (SP). Using Western blot analysis and immunofluorescence studies, we examined the expression levels of SP and its preferred G protein-coupled TK Receptor 1 (NK1R) and apoptosis markers in biopsies of the minor salivary gland (MSG) from pSS patients compared with patients with idiopathic sicca syndrome. We confirmed a quantitative decrease in the amount of SP in the MSG of pSS patients and demonstrated a significant increase in NK1R levels compared with sicca subjects, indicating the involvement of SP fibers and NK1R in the impaired salivary secretion observed in pSS patients. Moreover, the increase in apoptosis (PARP-1 cleavage) in pSS patients was shown to be related to JNK phosphorylation. Since there is no satisfactory therapy for the treatment of secretory hypofunction in pSS patients, the SP pathway may be a new potential diagnostic tool or therapeutic target

Wounds difficult to heal. An effective treatment strategy

OBJECTIVE: Treatment of wounds difficult to heal concerns 50% of the elderly population in Italy and is therefore a relevant social burden. The present study shows how the treatment with autologous leuco-platelets reduces the healing time of wounds improving the functional recovery. PATIENTS AND METHODS: Patients (n=100) with ulcers of the legs were divided in two groups: 1) 50 patients treated with conventional therapies; 2) 50 patients treated with autologous leuco-platelet concentrate (LPC) and hyaluronic acid (HIAFF, Hyalofill-F ® as a scaffold. RESULTS: After 2 months, a 49% reduction in wound area was observed in the second group and in about 65% wound reduction was achieved in 15 days (4 LPC dressings). In contrast, patients treated by conventional therapies, showed a longer healing time and a greater percentage of failures. Morphometric analysis of biopsy samples obtained from the edge as well as from the bottom of the lesions obtained from the LPC group, detected an abundant presence of neoformed capillaries, characterized by a cubic, "reactive endothelium", close to the site of LPC infiltration. CONCLUSIONS: These results suggest that healing was promoted not only by limiting bacterial infections but also by the release of chemotactic and proangiogenic factors from leukocytes and platelets, improving the neoformation of capillaries.Abstract: Objective: Treatment of wounds difficult to heal concerns 50% of the elderly population in Italy and is therefore a relevant social burden. The present study shows how the treatment with autologous leuco-platelets reduces the healing time of wounds improving the functional recovery. Patients and Methods: Patients (n=100) with ulcers of the legs were divided in two groups: 1) 50 patients treated with conventional therapies; 2) 50 patients treated with autologous leuco-platelet concentrate (LPC) and hyaluronic acid (HIAFF, Hyalofill-F®) as a scaffold. Results: After 2 months, a 49% reduction in wound area was observed in the second group and in about 65% wound reduction was achieved in 15 days (4 LPC dressings). In contrast, patients treated by conventional therapies, showed a longer healing time and a greater percentage of failures. Morphometric analysis of biopsy samples obtained from the edge as well as from the bottom of the lesions obtained from the LPC group, detected an abundant presence of neoformed capillaries, characterized by a cubic, "reactive endothelium", close to the site of LPC infiltration. Conclusions: These results suggest that healing was promoted not only by limiting bacterial infections but also by the release of chemotactic and proangiogenic factors from leukocytes and platelets, improving the neoformation of capillaries

Use of a new generation of capillary electrophoresis to quantify circulating free DNA in non-small cell lung cancer.

Circulating free DNA (cfDNA) is present in higher concentration in non-small-cell lung cancer (NSCLC) patients than in controls. This study was designed to assess the sensitivity and specificity of Agilent 2100 Bioanalyzer to identify patients with NSCLC and to compare it with quantitative RealTime-PCR (RT-qPCR) assay. 30 NSCLC patients and 26 controls were analyzed. The amount of cfDNA was determined both through quantitative RT-PCR targeting the human β-actin gene and by Agilent 2100 Bioanalyzer. Performances of the assays were calculated by the receiver operating characteristic (ROC) curves. The mean cfDNA concentration, obtained through the use of Agilent 2100 Bioanalyzer, in NSCLC patients (94.5. ng/mL) was almost twice the concentration detected in controls (42.8. ng/mL) as well as found by RT-qPCR (22.5. ng/mL vs 7.1. ng/mL, p. <. 0.001). The area under curve of Agilent 2100 Bioanalyzer and RT-PCR showed that there are no statistically significant differences between these tests (p. <. 0.92).This study shows that Agilent 2100 Bioanalyzer is an effective diagnostic tool to discriminate NSCLC patients from healthy individuals and suggests a new approach for early detection of NSCLC. © 2013 Elsevier B.V

Silencing of phosphoinositide-specific phospholipase C ε remodulates the expression of the phosphoinositide signal transduction pathway in human osteosarcoma cell lines.

Ezrin, a member of the ezrin-radixin-moesin family, is involved in the metastatic spread of osteosarcoma. Ezrin binds phosphatydil inositol-4,5-bisphosphate (PIP2), a crucial molecule of the phosphoinositide signal transduction pathway. PIP2 levels are regulated by phosphoinositide-specific phospholipase C (PI-PLC) enzymes. PI-PLCε isoform, a well-characterized direct effector of rat sarcoma (RAS), is at a unique convergence point for the broad range of signaling pathways that promote RAS GTPase-mediated signalling. Materials and Methods. By using molecular biology methods and microscopic analyses, we analyzed the expression of ezrin and PLC genes after silencing of PLCE (OMIM *608414) in 143B and Hs888 cell lines. The growth rate of the cells was slowed, and the expression of ezrin, PLCB1, PLCG2 and PLCD4 was significantly modified. Ezrin displacement from the plasma membrane was observed. The present results corroborate the hypothesis that ezrin and the PI signal transduction system are involved in a common network. Copyright© 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved

Comparison of phosphoinositide-specific phospholipase c panel of expression of human osteoblasts versus mg-63 and saos osteoblast-like cells

Background: A large number of phospholipase C (PLC) enzymes, both mRNA transcripts and proteins, have been detected in osteoblasts, corroborating the importance of calcium regulation in bone tissue. MG-63 and SaOS-2 human osteosarcoma cell lines are actually considered osteoblast-like cells, and are therefore widely used as experimental models for osteoblasts. Objectives: Our aim was to verify whether MG-63 or SaOS-2 cells might also represent appropriate experimental osteoblast models for signal transduction studies, with special regard to the phosphoinositide (PI) pathway. We analyzed the expression and the subcellular distribution of enzymes related to calcium signal transduction (the PI-specific PLC family), which are known to possess high cell/tissue specificity. Materials and Methods: The expression of PLC genes was analyzed by performing RT-PCR experiments. The presence of PLC enzymes and their subcellular distribution within the cells was analyzed with immunofluorescence experiments. Results: Osteoblasts, MG-63 cells, and SaOS-2 cells have expression panels similar to those of PLC enzymes. However, slight differences were found in the expression of enzymes belonging to the PLC subfamily. Conclusions: MG-63 and SaOS-2 osteosarcoma cell lines might not represent appropriate experimental models for studies that aim to analyze signal transduction in osteoblasts