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Different Signaling Pathways (different + signaling_pathway)
Selected AbstractsEffects of Fibronectin, VEGF and Angiostatin on the Expression of MMPs through Different Signaling Pathways in the JEG-3 CellsAMERICAN JOURNAL OF REPRODUCTIVE IMMUNOLOGY, Issue 4 2003Jian Zhang Problem: The objective of this study was to evaluate the possible signal pathway of fibronectin (FN), vascular endothelial growth factor (VEGF) and angiostatin (AS) on the expression of matrix metalloproteinases (MMPs) in JEG-3 cells. Methods of study: JEG-3 cells were cultured and were examined for the effect of FN, VEGF and AS on the expression of MMPs by immunocytochemistry, gelatin zymography, Western blot analysis and reverse transcription-polymerase chain reaction (RT-PCR). Results: We found that up-regulation of the expression of MMPs was induced by FN and VEGF through the focal adhesion kinase (FAK)/mitogen-activated protein kinase (MAPK) and Flt-1/p38SAPK/MAPKAPK2 signaling pathways, respectively. Furthermore, AS down-regulated the expression of MMPs through the integrin ,V,3/FAK signaling pathway independent of the integrin-binding motif Arg-Gly-Asp (RGD). Conclusion: These data indicate that the expression of MMPs is regulated by many independent factors (such as FN, VEGF and AS) through different signaling pathways which influence the behavior of trophoblast cells. [source] Different signaling pathways in the livers of patients with chronic hepatitis B or chronic hepatitis C,HEPATOLOGY, Issue 5 2006Masao Honda The clinical manifestations of chronic hepatitis B (CH-B) and chronic hepatitis C (CH-C) are different. We previously reported differences in the gene expression profiles of liver tissue infected with CH-B or CH-C; however, the signaling pathways underlying each condition have yet to be clarified. Using a newly constructed cDNA microarray consisting of 9614 clones selected from 256,550 tags of hepatic serial analysis of gene expression (SAGE) libraries, we compared the gene expression profiles of liver tissue from 24 CH-B patients with those of 23 CH-C patients. Laser capture microdissection was used to isolate hepatocytes from liver lobules and infiltrating lymphoid cells from the portal area, from 16 patients, for gene expression analysis. Furthermore, the comprehensive gene network was analyzed using SAGE libraries of CH-B and CH-C. Supervised and nonsupervised learning methods revealed that gene expression was correlated more with the infecting virus than any other clinical parameters such as histological stage and disease activity. Pro-apoptotic and DNA repair responses were predominant in CH-B with p53 and 14-3-3 interacting genes having an important role. In contrast, inflammatory and anti-apoptotic phenotypes were predominant in CH-C. These differences would evoke different oncogenic factors in CH-B and CH-C. In conclusion, we describe the different signaling pathways induced in the livers of patients with CH-B or CH-C. The results might be useful in guiding therapeutic strategies to prevent the development of hepatocellular carcinoma in cases of CH-B and CH-C. (HEPATOLOGY 2006;44:1122,1138.) [source] Altered integrin mechanotransduction in human nucleus pulposus cells derived from degenerated discsARTHRITIS & RHEUMATISM, Issue 2 2009Christine Lyn Le Maitre Objective Several studies have demonstrated biologic responses of intervertebral disc (IVD) cells to loading, although the mechanotransduction pathways have not been elucidated. In articular chondrocytes, which have a phenotype similar to that of IVD cells, a number of mechanoreceptors have been identified, with ,5,1 integrin acting as a predominant mechanoreceptor. The purpose of this study was to investigate the role of integrin signaling in IVD cells during mechanical stimulation and to determine whether RGD integrins are involved. Methods Human nucleus pulposus (NP) cells derived from nondegenerated and degenerated discs were subjected to dynamic compressive loading in the presence of an RGD inhibitory peptide. Expression of the ,5,1 heterodimer in IVD tissue was examined by immunohistochemistry and possible alternative mechanoreceptors by real-time quantitative polymerase chain reaction. Results Aggrecan gene expression was decreased following loading of NP cells from nondegenerated and degenerated discs. This response was inhibited by treatment with an RGD peptide in cells from nondegenerated, but not degenerated, IVDs. Immunohistochemistry demonstrated that expression of the ,5,1 heterodimer was unaltered in degenerated IVD tissue as compared with normal IVD tissue. Conclusion Our results indicate that the mechanotransduction pathways are altered in cells from degenerated IVDs. Mechanosensing in NP cells from nondegenerated discs occurs via RGD integrins, possibly via the ,5,1 integrin, while cells from degenerated discs show a different signaling pathway that does not appear to involve RGD integrins. [source] IL-27 controls the development of inducible regulatory T cells and Th17 cells via differential effects on STAT1EUROPEAN JOURNAL OF IMMUNOLOGY, Issue 7 2007Clemens Neufert Abstract IL-27 is an IL-12-related cytokine frequently present at sites of inflammation that can promote both anti- and pro-inflammatory immune responses. Here, we have analyzed the mechanisms how IL-27 may drive such divergent immune responses. While IL-27 suppressed the development of proinflammatory Th17 cells, a novel role for this cytokine in inhibiting the development of anti-inflammatory, inducible regulatory T cells (iTreg) was identified. In fact, IL-27 suppressed the development of adaptive, TGF-,-induced Forkhead box transcription factor p3-positive (Foxp3+) Treg. Whereas the blockade of Th17 development was dependent on the transcription factor STAT1, the suppression of iTreg development was STAT1 independent, suggesting that IL-27 utilizes different signaling pathways to shape T cell-driven immune responses. Our data thus demonstrate that IL-27 controls the development of Th17 and iTreg cells via differential effects on STAT1. [source] Transient expression of endothelins in the amoeboid microglial cells in the developing rat brainGLIA, Issue 6 2006Chun-Yun Wu Abstract Amoeboid microglial cells (AMC) which transiently exist in the corpus callosum in the postnatal rat brain expressed endothelins (ETs), specifically endothelin-1 (ET-1) and ET3 as revealed by real time RT-PCR. ET immunoreactive AMC occurred in large numbers at birth, but were progressively reduced with age and were undetected in 14 days. In rats subjected to hypoxia exposure, ET immunoexpression in AMC was reduced but the incidence of apoptotic cells was not increased when compared with the control suggesting that this was due to its downregulation that may help regulate the constriction of blood vessels bearing ET-A receptor. AMC were endowed ET-B receptor indicating that ET released by the cells may also act via an autocrine manner. In microglia activated by lipopolysaccharide (LPS), ET-1 mNA expression coupled with that of monocyte chemoattractant protein (MCP-1) and stromal derived factor-1 (SDF-1) was markedly increased; ET-3 mRNA, however, remained unaffected. AMC exposed to oxygen glucose deprivation (OGD) in vitro resulted in increase in both ET-1 and ET-3 mRNA expression. It is suggested that the downregulated ETs expression in vivo of AMC subjected to hypoxia as opposed to its upregulated expression in vitro may be due to the complexity of the brain tissue. Furthermore, the differential ET-1 and ET-3 mRNA expression in LPS and OGD treatments may be due to different signaling pathways independently regulating the two isoforms. The present novel finding has added microglia as a new cellular source of ET that may take part in multiple functions including regulating vascular constriction and chemokines release. © 2006 Wiley-Liss, Inc. [source] Different signaling pathways in the livers of patients with chronic hepatitis B or chronic hepatitis C,HEPATOLOGY, Issue 5 2006Masao Honda The clinical manifestations of chronic hepatitis B (CH-B) and chronic hepatitis C (CH-C) are different. We previously reported differences in the gene expression profiles of liver tissue infected with CH-B or CH-C; however, the signaling pathways underlying each condition have yet to be clarified. Using a newly constructed cDNA microarray consisting of 9614 clones selected from 256,550 tags of hepatic serial analysis of gene expression (SAGE) libraries, we compared the gene expression profiles of liver tissue from 24 CH-B patients with those of 23 CH-C patients. Laser capture microdissection was used to isolate hepatocytes from liver lobules and infiltrating lymphoid cells from the portal area, from 16 patients, for gene expression analysis. Furthermore, the comprehensive gene network was analyzed using SAGE libraries of CH-B and CH-C. Supervised and nonsupervised learning methods revealed that gene expression was correlated more with the infecting virus than any other clinical parameters such as histological stage and disease activity. Pro-apoptotic and DNA repair responses were predominant in CH-B with p53 and 14-3-3 interacting genes having an important role. In contrast, inflammatory and anti-apoptotic phenotypes were predominant in CH-C. These differences would evoke different oncogenic factors in CH-B and CH-C. In conclusion, we describe the different signaling pathways induced in the livers of patients with CH-B or CH-C. The results might be useful in guiding therapeutic strategies to prevent the development of hepatocellular carcinoma in cases of CH-B and CH-C. (HEPATOLOGY 2006;44:1122,1138.) [source] PTHrP Signaling Targets Cyclin D1 and Induces Osteoblastic Cell Growth Arrest,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 6 2005Nabanita S Datta PhD Abstract PTHrP control of the MC3T3-E1 cell cycle machinery showed that, during differentiation, PTHrP induced G1 growth arrest. Cyclin D1 was a critical mediator as a downstream effector of cAMP, PKC, and MAPK signaling, and the process was PKA-independent. The involvement of JunB has been found critical for PTHrP effects. Introduction: PTH-related protein (PTHrP) has been implicated in the control of bone cell turnover, but the mechanisms underlying its effect on osteoblast proliferation and differentiation have not been clearly defined. The mechanisms by which PTHrP impacts cell cycle proteins and the role of signaling pathways in differentiated osteoblasts were studied. Materials and Methods: To elucidate the role of PTHrP, flow cytometric analyses were performed using MC3T3-E1 and primary mouse calvarial cells. Relative protein abundance (Western blot), physical association of partners (immunoprecipitation), and kinase activities (in vitro kinase assays using either GST-Rb or H1-histone as substrates) of cell cycle-associated proteins in vehicle and PTHrP-treated 7-day differentiated cells were determined. ELISA and/or Northern blot analyses were done to evaluate JunB and cyclin D1 expression. SiRNA-mediated gene silencing experiments were performed to silence JunB protein. Finally, inhibitors of cAMP, protein kinase A (PKA), protein kinase C (PKC), and mitogen-activated protein kinase (MAPK) were used to determine involvement of different signaling pathways. Results: PTHrP inhibited cyclin D1 protein expression 7-fold in a dose- and time-dependent manner and increased the level of p16 protein in differentiated osteoblasts. Additionally, PTHrP reduced cyclin D1-CDK4/CDK6 and CDK1 kinase activities. Forskolin, a cAMP agonist, mimicked PTHrP action, and the PKC inhibitor, GF109203X, slightly blocked downregulation of cyclin D1, implying involvement of both cAMP and PKC. U0126, a MAPK inhibitor, alone decreased cyclin D1 protein, suggesting that the basal cyclin D1 protein is MAPK dependent. H-89, a PKA inhibitor, did not alter the effect of PTHrP on cyclin D1, suggesting a PKA-independent mechanism. Finally, expression of JunB, an activating protein-1 transcription factor, was significantly upregulated, and silencing JunB (siRNA) partially reversed the cyclin D1 response, implying involvement of JunB in the PTHrP-mediated growth arrest of MC3T3-E1 cells. Conclusion: PTHrP upregulates JunB and reduces cyclin D1 expression while inducing G1 cell cycle arrest in differentiated osteoblasts. Such regulation could be an important determinant of the life span and bone-forming activity of osteoblasts. [source] Induction of Transcriptional Activity of the Cyclic Adenosine Monophosphate Response Element Binding Protein by Parathyroid Hormone and Epidermal Growth Factor in Osteoblastic Cells,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 8 2002John T. Swarthout Abstract Previously, we have shown that parathyroid hormone (PTH) transactivation of cyclic adenosine monophosphate (cAMP) response element binding protein (CREB) requires both serine 129 (S129) and serine 133 (S133) in rat osteosarcoma cells UMR 106-01 (UMR) cells. Furthermore, although protein kinase A (PKA) is responsible for phosphorylation at S133, glycogen synthase kinase 3, (GSK-3,) activity is required and may be responsible for phosphorylation of CREB at S129. Here, we show, using the GAL4-CREB reporter system, that epidermal growth factor (EGF) can transactivate CREB in UMR cells in addition to PTH. Additionally, treatment of UMR cells with both PTH and EGF results in greater than additive transactivation of CREB. Furthermore, using mutational analysis we show that S129 and S133 are required for EGF-induced transcriptional activity. EGF activates members of the MAPK family including p38 and extracellular signal,activated kinases (ERKs), and treatment of UMR cells with either the p38 inhibitor (SB203580) or the MEK inhibitor (PD98059) prevents phosphorylation of CREB at S133 by EGF but not by PTH. Treatment of cells with either SB203580 or PD98059 alone or together significantly inhibits transactivation of CREB by EGF but not by PTH, indicating that EGF regulates CREB phosphorylation and transactivation through p38 and ERKs and PTH does not. Finally, the greater than additive transactivation of CREB by PTH and EGF is significantly inhibited by the PKA inhibitor H-89 or by cotreatment with SB203580 and PD98059. Thus, several different signaling pathways in osteoblastic cells can converge on and regulate CREB activity. This suggests, in vivo, that circulating agents such as PTH and EGF are acting in concert to exert their effects. [source] Synergistic antiviral effect of a combination of mouse interferon-, and interferon-, on mouse hepatitis virusJOURNAL OF MEDICAL VIROLOGY, Issue 2 2003Uichiro Fuchizaki Abstract Although interferon (IFN)-, and IFN-, have been reported to exhibit a synergistic antiviral effect through the different signaling pathways in vitro, their therapeutic efficacy is not well defined in vivo. The current study was carried out to investigate the combined antiviral effect in a model of mouse hepatitis virus Type 2 (MHV-2) infection, in which fulminant hepatitis is developed. MHV-2 was injected intraperitoneally into 4-week-old ICR mice, IFN or the vehicle was administered intramuscularly for 5 days, and the antiviral effect was evaluated based on survival periods, liver histology, serum alanine transaminase (ALT) levels, and MHV-2 virus titers in the liver tissues. The animals in the group treated with a combination of IFN-, and IFN-, survived for longer periods than the groups treated with IFN-, alone and IFN-, alone (IFN-, 103 (IU/mouse)/-, 103 vs. IFN-, 103, P,<,0.005; IFN-, 103/-, 103 vs. IFN-, 103, P,<,0.001). This is consistent with the lower levels of hepatocellular necrosis and serum ALT and the decreased titers of MHV-2 virus in the liver tissues (48 hr, P,<,0.001; 72 hr, P,<,0.001). These findings indicate that a combination of IFN-, and IFN-, exhibits a synergistic antiviral effect on MHV-2 infection. The biology of MHV-2 is quite different from that of human hepatitis viruses; however, these results suggest the beneficial combined therapy of IFN-, and IFN-, for the treatment of human viral hepatitis. J. Med. Virol. 69:188,194, 2003. © 2003 Wiley-Liss, Inc. [source] Regulated trafficking of neurotransmitter transporters: common notes but different melodiesJOURNAL OF NEUROCHEMISTRY, Issue 1 2002Michael B. Robinson Abstract The activity of biogenic amine and amino acid neurotransmitters is limited by presynaptic and astrocytic Na+ -dependent transport systems. Their functional importance is underscored by the observation that these transporters are the targets of broad classes of psychotherapeutic agents, including antidepressants and stimulants. Early studies suggested that the activity of these transporters can be fine tuned by a number of different signaling pathways. In the past five years, several groups have provided compelling evidence that changing the cell surface availability of these transporters contributes to this fine tuning. This regulated trafficking can result in rapid (within minutes) increases or decreases in the plasma membrane expression of these transporters and is independent of transcriptional or translational control mechanisms. Many of the same signaling molecules, including protein kinase C (PKC), tyrosine kinase, phosphatidylinositol 3-kinase (P13-K), and protein phosphatase, regulate the transporters for different neurotransmitters. In addition to these classical receptor activated pathways, transporter substrates also regulate activity and cell surface expression of these transporters. In fact, some of the transporters form complexes with signaling molecules. Given the functional and genetic similarities of these transporters, it is not surprising that the same signaling molecules regulate their trafficking, but except for the molecules, the actual effects on individual transporters are remarkably different. It,is as if the same musical notes have been rearranged into several different melodies. [source] Effects of porcine 25 kDa amelogenin and its proteolytic derivatives on bone sialoprotein expressionJOURNAL OF PERIODONTAL RESEARCH, Issue 5 2010Y. Nakayama Nakayama Y, Yang L, Mezawa M, Araki S, Li Z, Wang Z, Sasaki Y, Takai H, Nakao S, Fukae M, Ogata Y. Effects of porcine 25 kDa amelogenin and its proteolytic derivatives on bone sialoprotein expression. J Periodont Res 2010; 45: 602,611. © 2010 John Wiley & Sons A/S Background and Objective:, Amelogenins are hydrophobic proteins that are the major component of developing enamel. Enamel matrix derivative has been used for periodontal regeneration. Bone sialoprotein is an early phenotypic marker of osteoblast differentiation. In this study, we examined the ability of porcine amelogenins to regulate bone sialoprotein transcription. Material and Methods:, To determine the molecular basis of the transcriptional regulation of the bone sialoprotein gene by amelogenins, we conducted northern hybridization, transient transfection analyses and gel mobility shift assays using the osteoblast-like ROS 17/2.8 cells. Results:, Amelogenins (100 ng/mL) up-regulated bone sialoprotein mRNA at 3 h, with maximal mRNA expression occurring at 12 h (25 and 20 kDa) and 6 h (13 and 6 kDa). Amelogenins (100 ng/mL, 12 h) increased luciferase activities in pLUC3 (nucleotides ,116 to +60), and 6 kDa amelogenin up-regulated pLUC4 (nucleotides ,425 to +60) activity. The tyrosine kinase inhibitor inhibited amelogenin-induced luciferase activities, whereas the protein kinase A inhibitor abolished 25 kDa amelogenin-induced bone sialoprotein transcription. The effects of amelogenins were abrogated by 2-bp mutations in the fibroblast growth factor 2 response element (FRE). Gel-shift assays with radiolabeled FRE, homeodomain-protein binding site (HOX) and transforming growth factor-beta1 activation element (TAE) double-strand oligonucleotides revealed increased binding of nuclear proteins from amelogenin-stimulated ROS 17/2.8 cells at 3 h (25 and 13 kDa) and 6 h (20 and 6 kDa). Conclusion:, These results demonstrate that porcine 25 kDa amelogenin and its proteolytic derivatives stimulate bone sialoprotein transcription by targeting FRE, HOX and TAE in the bone sialoprotein gene promoter, and that full-length amelogenin and amelogenin cleavage products are able to regulate bone sialoprotein transcription via different signaling pathways. [source] Differential phosphorylation of myosin light chain (Thr)18 and (Ser)19 and functional implications in plateletsJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 10 2010T. M. GETZ Summary. Background:, Myosin IIA is an essential platelet contractile protein that is regulated by phosphorylation of its regulatory light chain (MLC) on residues (Thr)18 and (Ser)19 via the myosin light chain kinase (MLCK). Objective:, The present study was carried out to elucidate the mechanisms regulating MLC (Ser)19 and (Thr)18 phosphorylation and the functional consequence of each phosphorylation event in platelets. Results:, Induction of 2MeSADP-induced shape change occurs within 5 s along with robust phosphorylation of MLC (Ser)19 with minimal phosphorylation of MLC (Thr)18. Selective activation of G12/13 produces both slow shape change and comparably slow MLC (Thr)18 and (Ser)19 phosphorylation. Stimulation with agonists that trigger ATP secretion caused rapid MLC (Ser)19 phosphorylation while MLC (Thr)18 phosphorylation was coincident with secretion. Platelets treated with p160ROCK inhibitor Y-27632 exhibited a partial inhibition in secretion and had a substantial inhibition in MLC (Thr)18 phosphorylation without effecting MLC (Ser)19 phosphorylation. These data suggest that phosphorylation of MLC (Ser)19 is downstream of Gq/Ca2+ -dependent mechanisms and sufficient for shape change, whereas MLC (Thr)18 phosphorylation is substantially downstream of G12/13 -regulated Rho kinase pathways and necessary, probably in concert with MLC (Ser)19 phosphorylation, for full contractile activity leading to dense granule secretion. Overall, we suggest that the amplitude of the platelet contractile response is differentially regulated by a least two different signaling pathways, which lead to different phosphorylation patterns of the myosin light chain, and this mechanism results in a graded response rather than a simple on/off switch. [source] Effects of Fibronectin, VEGF and Angiostatin on the Expression of MMPs through Different Signaling Pathways in the JEG-3 CellsAMERICAN JOURNAL OF REPRODUCTIVE IMMUNOLOGY, Issue 4 2003Jian Zhang Problem: The objective of this study was to evaluate the possible signal pathway of fibronectin (FN), vascular endothelial growth factor (VEGF) and angiostatin (AS) on the expression of matrix metalloproteinases (MMPs) in JEG-3 cells. Methods of study: JEG-3 cells were cultured and were examined for the effect of FN, VEGF and AS on the expression of MMPs by immunocytochemistry, gelatin zymography, Western blot analysis and reverse transcription-polymerase chain reaction (RT-PCR). Results: We found that up-regulation of the expression of MMPs was induced by FN and VEGF through the focal adhesion kinase (FAK)/mitogen-activated protein kinase (MAPK) and Flt-1/p38SAPK/MAPKAPK2 signaling pathways, respectively. Furthermore, AS down-regulated the expression of MMPs through the integrin ,V,3/FAK signaling pathway independent of the integrin-binding motif Arg-Gly-Asp (RGD). Conclusion: These data indicate that the expression of MMPs is regulated by many independent factors (such as FN, VEGF and AS) through different signaling pathways which influence the behavior of trophoblast cells. [source] Specific cleavage of ribosomal RNA and mRNA during victorin-induced apoptotic cell death in oatTHE PLANT JOURNAL, Issue 6 2006Trinh X. Hoat Summary Here we report that rRNA and mRNA are specifically degraded in oat (Avena sativa L.) cells during apoptotic cell death induced by victorin, a host-selective toxin produced by Cochliobolus victoriae. Northern analysis indicated that rRNA species from the cytosol, mitochondria and chloroplasts were all degraded via specific degradation intermediates during victorin-induced apoptotic cell death but, in contrast, they were randomly digested in necrotic cell death induced by 30 mm CuSO4 and heat shock. This indicates that specific rRNA cleavage could be controlled by an intrinsic program. We also observed specific cleavage of mRNA of housekeeping genes such as actin and ubiquitin during victorin-induced cell death. Interestingly, no victorin-induced mRNA degradation was detected with stress-responding genes such as PR-1, PR-10 and GPx throughout the experimental period. The RNA degradation mostly, but not always, occurred in parallel with DNA laddering, but pharmacological studies indicated that these processes are regulated by different signaling pathways with some overlapping upstream signals. [source] A novel cold-inducible gene from Arabidopsis, RCI3, encodes a peroxidase that constitutes a component for stress toleranceTHE PLANT JOURNAL, Issue 1 2002Francisco Llorente Summary A cDNA from Arabidopsis corresponding to a new cold-inducible gene, RCI3 (for Rare Cold Inducible gene 3), was isolated. Isoelectric focusing electrophoresis and staining of peroxidase activity demonstrated that RCI3 encodes an active cationic peroxidase. RNA-blot analysis revealed that RCI3 expression in response to low temperature is negatively regulated by light, as RCI3 transcripts were exclusively detected in etiolated seedlings and roots of adult plants. RCI3 expression was also induced in etiolated seedlings, but not in roots, exposed to dehydration, salt stress or ABA, indicating that it is subjected to a complex regulation through different signaling pathways. Analysis of transgenic plants containing RCI3::GUS fusions established that this regulation occurs at the transcriptional level during plant development, and that cold-induced RCI3 expression in roots is mainly restricted to the endodermis. Plants overexpressing RCI3 showed an increase in dehydration and salt tolerance, while antisense suppression of RCI3 expression gave dehydration- and salt-sensitive phenotypes. These results indicate that RCI3 is involved in the tolerance to both stresses in Arabidopsis, and illustrate that manipulation of RCI3 has a potential with regard to plant improvement of stress tolerance. [source] Contribution of death receptor and mitochondrial pathways to Fas-mediated apoptosis in the prostatic carcinoma cell line PC3THE PROSTATE, Issue 4 2002Natalya V. Guseva Abstract BACKGROUND Two main pathways of apoptosis in mammalian cells have been described: the death receptor pathway and the mitochondrial pathway. Two different cell types have been identified for Fas-mediated apoptosis, each using almost exclusively one of two different signaling pathways. Human prostatic carcinoma cell line, PC3 is sensitive to Fas-mediated apoptosis, but relation of receptor and mitochondrial pathways is not clear. METHODS Cell viability was estimated by calcein assay. Apoptosis was determined by preparation of DNA ladder. Expression of Fas-associated death domain-dominant negative (FADD-DN) and Bcl-2, activation of caspases, PARP, DFF45, Bid cleavage, and cytochrome c release were assessed using Western blotting techniques. [35S] Methionine-labeled caspase-3 was transcribed in vitro and translated using the TNT kit (Promega). A vector containing caspase-3 was prepared by the ligation of EcoR I/BamHI flanked PCR fragment of full size caspase-3 cDNA into pBlusckript II SK(+/,) (Stratagen). RESULTS Overexpression of both FADD-DN and Bcl-2 genes prevent Fas-mediated apoptosis in PC3. As predicted, overexpression of FADD-DN prevented activation of caspase-8 and Bid cleavage and attenuated the release of cytochrome c and activation of caspases -2, -7, and -9. Bcl-2 overexpression did not affect caspase-8 activation and cleavage of Bid but blocked the release of cytochrome c and activation of mitochondria localized caspases -2, -7, and,9. Overexpression of FADD-DN and Bcl-2 affected the activation of caspase-3 and PARP cleavage differently: FADD-DN attenuated the activation of caspase-3 and PARP cleavage whereas Bcl-2 overexpression prevented caspase-3 activation and completely blocked cleavage of PARP. CONCLUSIONS These data suggest that activation of caspase-8 is necessary but not sufficient to complete Fas-mediated apoptosis in PC3 cells without activation of the mitochondrial pathway. In addition, caspase-3 activation after Fas-receptor ligation involves two steps and is dependent on mitochondrial activation. Prostate 51: 231,240, 2002. © 2002 Wiley-Liss, Inc. [source] Signaling pathways in innate immunityACTA OPHTHALMOLOGICA, Issue 2008A SALMINEN Inflammation has a key role in the pathogenesis of AMD. This lecture will review the recent progress in understanding the different host-defence mechanisms against pathogens and self-based danger signals involved in the activation of innate immunity. The innate defence system utilizes pattern recognition receptors (PRR) to respond to a variety of pathogen-associated (PAMP) and danger-associated (DAMP) molecular structures. Along with the well-known complement and scavenger receptor systems, Toll-like receptors (TLR) and NOD-like receptors (NLR) have also a crucial part in host-defence and these receptor systems can be activated both by PAMPs and DAMPs. Pattern recognition receptors are located either in cell surface, such as TLR2 and TLR4, or in intracellular locations, e.g. TLR3, TLR9 and all NLRs. PRRs show some specificity to ligands and also in downstream they activate different signaling pathways, most common of which are NF-kB and IRF-dependent pathways inducing inflammatory responses. Retinal pigment epithelial cells (RPE) have an important role in eye host-defence, both at apical and basolateral surfaces. Most of the TLRs are expressed in RPE cells, especially TLR3 and TLR4, and they can participate in photoreceptor outer segment recognition. TLR3 can also suppress angiogenesis. The functions of NLRs, e.g. those forming inflammasomes, are still unknown, although the danger-type of activation signals, such as oxidative stress and potassium efflux, are present in retinal pigment epithelium. It seems that the activation of innate immunity system via DAMPs and PRRs may have a central role in the pathogenesis of AMD. [source] | |||||||||||||