Activation of focal adhesion kinase enhances the adhesion and invasion of pancreatic cancer cells via extracellular signal-regulated kinase-1/2 signaling pathway activation.
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BACKGROUND: Interaction with integrin and focal adhesion kinase (FAK) regulates the cancer cell adhesion and invasion into extracellular matrix (ECM). In addition, phosphorylation of FAK correlates with the increase of cell motility and invasion. Adhesion and spreading of cancer cells on a variety of ECM proteins, including collagen type IV (Coll IV), leads to an increase in tyrosine phosphorylation and activation of FAK. In this study, we investigated the mechanism of activation of FAK and its downstream extracellular signal-regulated kinase (ERK)-1/2 signaling following stimulation by interleukin (IL)-1alpha and adhesion to ECM with subsequent enhancement of pancreatic cancer cell adhesion and invasion. RESULTS: In immunoblotting analysis, all three pancreatic cancer cell lines (AsPC-1, BxPC-3, and Capan-2) expressed the protein of FAK and beta1 integrin. Enhancement of FAK protein association with beta1 integrin when cells were plated on Coll IV was more increased by stimulation with IL-1alpha. Preincubation with anti-beta1 integrin antibody and FAK siRNA transfection inhibited the association of FAK with beta1 integrin of pancreatic cancer cells. FAK phosphorylation was observed by adhesion to Coll IV, furthermore, stronger FAK phosphorylation was observed by stimulation with IL-1alpha of pancreatic cancer cells adhered to Coll IV in time-dependent manner. Genistein, a tyrosine kinase inhibitor, markedly inhibited the FAK phosphorylation. IL-1alpha stimulation and Coll IV adhesion enhanced the activation of Ras, as evidenced by the increased Ras-GTP levels in pancreatic cancer cells. Activation of Ras correlated with the phosphorylation of ERK. While not statistical affecting the apoptosis of pancreatic cancer cells, IL-1alpha-induced adhesion and invasion on Coll IV were inhibited with FAK gene silencing by siRNA, beta1 integrin blocking, and inhibition of FAK phosphorylation. PD98059, a MEK inhibitor, also inhibited IL-1alpha-induced enhancement of adhesion and invasion in pancreatic cancer cells. CONCLUSION: Our results demonstrated that activation of FAK is involved with the aggressive capability in pancreatic cancer through Ras/ERK signaling pathway. Based on our results, we suggest that the modification of IL-1, FAK, and integrins functions might be a novel therapeutic approach to aggressive spread of pancreatic cancer. (+info)
Immune phenomena involved in the in vivo regression of fibrosarcoma cells expressing cell-associated IL-1alpha.
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Constitutive expression of cell-associated, but not secreted, interleukin-1alpha (IL-1alpha) by oncogene-transformed fibrosarcoma cells induced regressing tumors in mice, a phenomenon that was abrogated by the IL-1 inhibitor, the IL-1 receptor antagonist (IL-1Ra). On the contrary, non-IL-1alpha-expressing tumor cells induce progressive tumors in mice. In vivo and ex vivo experiments have shown that regression of IL-1alpha-positive fibrosarcoma cells depends on CD8(+) T cells, which can also be activated in CD4(+) T cell-depleted mice, with some contribution of natural killer cells. In spleens of mice bearing the non-IL-1alpha-expressing fibrosarcoma cells, some early and transient manifestations of antitumor-specific immunity, such as activation of specific proliferating T cells, are evident; however, no development of cytolytic T lymphocytes or other antitumor protective cells could be detected. In spleens of mice bearing the non-IL-1alpha-expressing fibrosarcoma cells, the development of early tumor-mediated suppression was observed, and in spleens of mice injected with IL-1alpha-positive fibrosarcoma cells, protective immunity developed in parallel to tumor regression. Treatment of mice bearing violent fibrosarcoma tumors with syngeneic-inactivated, IL-1alpha-positive fibrosarcoma cells, at a critical interval after injection of the malignant cells (Days 5-12), induced tumor regression, possibly by potentiating and amplifying transient antitumor cell immune responses or by ablation of tumor-mediated suppression. Membrane-associated IL-1alpha may thus serve as an adhesion molecule, which allows efficient cell-to-cell interactions between the malignant and immune effector cells that bear IL-1Rs and function as a focused cytokine with adjuvant activities at nontoxic, low levels of expression. Our results also point to the potential of using antitumor immunotherapeutic approaches using cell-associated IL-1alpha. (+info)
Signs of inflammation in both symptomatic and asymptomatic muscles from patients with polymyositis and dermatomyositis.
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OBJECTIVES: To determine whether muscle weakness is correlated with inflammation, expression of interleukin 1alpha (IL1alpha) and major histocompatibility complex (MHC) class I and II antigens on muscle fibres. METHODS: Biopsy specimens from clinically symptomatic (proximal muscles) and asymptomatic (all distal but two proximal) muscles in eight patients with polymyositis, three patients with dermatomyositis and six healthy controls were analysed by immunohistochemistry for the presence of T cells and macrophages, and expression of IL1alpha and of MHC class I and II antigens. RESULTS: were evaluated by conventional light microscopy and by computerised image analysis. Results: Inflammatory infiltrates with T cells and macrophages were observed to an equal degree in both symptomatic and asymptomatic muscle. The numbers of capillaries with IL1alpha expression were significantly higher (p<0.05) in the symptomatic and asymptomatic muscles of patients than in controls. The total IL1alpha expression per tissue section assessed by computerised image analysis was significantly higher in symptomatic muscles but not in asymptomatic muscles compared with that in controls. Neither the number of IL1alpha-positive capillaries nor the total IL1alpha expression differed significantly between symptomatic and asymptomatic muscles. Expression of MHC class I and II antigens on muscle fibres was detected in both symptomatic and asymptomatic muscles but rarely in healthy controls. CONCLUSIONS: Presence of inflammatory infiltrates, T cells and macrophages, and expression of MHC class I and II antigens and of IL1alpha on muscle fibres were independent of clinical symptoms, and were present to an equal degree in both proximal and distal muscles. Thus, other factors seem to determine the development of clinical symptoms. One such factor could be variations in physical demands. (+info)
Interleukin-1-induced NF-kappaB recruitment to the oxytocin receptor gene inhibits RNA polymerase II-promoter interactions in cultured human myometrial cells.
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The myometrial oxytocin receptor (OTR) is highly regulated during pregnancy, reaching maximal concentrations near term. These levels are then abruptly reduced in advanced labour and the post-partum period. Our goal was to examine the molecular basis for this reduction, using chromatin immunoprecipitation (ChIP). Interleukin-1alpha (IL1A) treatment of cultured human myometrial cells has previously been shown to reduce steady-state levels of OTR mRNA. We show further that IL1A reduced RNA polymerase II cross-linking to the otr promoter, as reflective of transcriptional inhibition. IL1A also increased the recruitment of nuclear factor kappaB (NF-kappaB) to a site 955 bp upstream from the transcriptional start site. Inhibition of NF-kappaB activation negated the effects of IL1A on polymerase II dissociation, indicating a causal relationship, at least in part, between recruitment of NF-kappaB and detachment of polymerase from the otherwise constitutively active otr promoter. IL1A treatment also resulted in increased histone H4 acetylation in the otr promoter region. Whereas NF-kappaB recruitment and histone acetylation are generally associated with activation of gene expression, our findings show that both processes can be involved in dissociation of RNA polymerase II from an active promoter. The results of these studies suggest that the elevation of IL1 in the myometrium occurring at the end of pregnancy initiates the process of down-regulation of OTRs in advanced labour, resulting in the desensitization of the myometrium to elevated levels of OT in the blood during lactation. (+info)
Interleukin 10 and transforming growth factor beta contribute to the development of experimentally induced allergic conjunctivitis in mice during the effector phase.
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AIM: To investigate the involvement of interleukin (IL)10 and transforming growth factor (TGF) beta in the development of experimentally induced allergic conjunctivitis in mice. METHODS: Balb/c mice were actively sensitised with ragweed in alum, and then challenged with ragweed in eye drops after 10 days. 24 h later, the conjunctivas, spleens and blood were collected for histological and cytokine expression analyses, proliferation and cytokine production assays and measurement of immunoglobulin (Ig) levels. Mice developing experimentally induced allergic conjunctivitis were injected intraperitoneally with 200 microg of anti-IL10 or anti-TGF beta antibodies at 0, 2, 4, 6 and 8 days (induction phase treatment) or 500 microg of antibodies 2 h before ragweed challenge (effector phase treatment). Normal rat IgG was used for control injections. RESULTS: Treatment with either anti-IL10 or anti-TGF beta antibodies during the induction phase did not affect eosinophil infiltration into the conjunctiva. By contrast, treatment with either antibody during the effector phase suppressed infiltration. During the effector phase, treatment with anti-TGF beta antibody, but not the anti-IL10 antibody, markedly up regulated proliferation and Th2 cytokine production by splenocytes. IL1alpha levels in the conjunctiva were reduced after treatment with either antibody; in addition, eotaxin and tumour necrosis factor alpha levels were reduced after treatment with antibody to TGF beta. CONCLUSIONS: IL10 and TGF beta do not have immunosuppressive roles in the development of experimentally induced allergic conjunctivitis. Rather, they augment the infiltration of eosinophils into the conjunctiva during the effector phase of experimentally induced allergic conjunctivitis. (+info)
R5- and X4-HIV-1 use differentially the endometrial epithelial cells HEC-1A to ensure their own spread: implication for mechanisms of sexual transmission.
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The mechanism of viral transmission across the mucosal barrier is poorly understood. Using the endometrial epithelium-derived cell line HEC-1A, we found that the cells are capable of sequestering large numbers of HIV-1 particles but are refractory to cell-free viral infection. The removal of heparan sulfate moieties of cell-surface proteoglycans (HSPG) from the apical pole of HEC-1A accounted for at least 60% of both R5- and X4-HIV-1 attachment, showing their important implication in viral attachment. HEC-1A cells also have the capacity to endocytose a weak proportion of the attached virus and pass it along to underlying cells. Fucose, N-acetylglucosamine and mannosylated-residues inhibited the transcytosis of some virus isolates, suggesting that mannose receptors can be implicated on the both R5- and X4-HIV-1 transcytosis. The inhibition of HIV transcytosis by blocking CCR5 mAb suggests the implication of specific interaction between the viral gp120 and sulfated moiety of syndecans during the transcytosis of mostly R5- and X4-HIV-1. At the basolateral pole of HEC-1A, HSPG sequestered X4- and not R5-HIV-1, highlighting the important role of HEC-1A as an X4 virus reservoir. The cell-free virus particles that have transcytosed could infect activated T cells but with a weaker efficiency than virus that had not transcytosed. The specific stimulation of HEC-1A by R5-HIV-1 increased the release of monocytes/chemokines-attracting chemokines (IL-8 and GR0) and proinflammatory cytokines (TNF-beta and IL-1alpha) that enhanced the production of virus by activated T cells. This study suggests that R5 and X4 viruses can differentially use epithelial cells to ensure their own spread. (+info)
Induction of the CXCL1 (KC) chemokine in mouse astrocytes by infection with the murine encephalomyelitis virus of Theiler.
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In the present study, we focused on the production of the chemokine CXCL1, also termed KC, by cultured Theiler murine encephalomyelitis virus (TMEV)-infected mouse astrocytes. cRNA from mock- and TMEV-infected cells was hybridized to the Affymetrix murine genome U74v2 DNA microarray. Hybridization data analysis demonstrated upregulation of two sequences coding for IL-8 and related to the GRO 1 oncogene MGSA. The murine counterpart of the above human genes has been reported to be the chemokine CXCL1 or KC, and therefore we studied its regulation, confirming its mRNA increase by Northern blots. The presence of CXCL1 in the supernatants of infected cells was further demonstrated by a specific ELISA and its intracellular accumulation by flow cytometry. This secreted CXCL1 was biologically active in a non species-specific way as it induces chemoattraction on human neutrophils and monocyte/macrophages, but not on CD3 positive lymphocytes. Its induction does not follow the MAP kinase pathway which transcripts are decrease in infected cells compared with uninfected astrocytes. Two inflammatory cytokines, IL-1alpha and TNF-alpha, which are also induced by TMEV in astrocytes, were potent inducers of CXCL1. Nevertheless, both mechanisms of induction follow different pathways as antibodies to both cytokines fail to inhibit TMEV-induced CXCL1 upregulation. Spinal cords but not brains from TMEV-infected SJL/J animals contain CXCL1 at the start of clinical signs of the disease. As no CXCL1 induction can be detected neither in cultured BALB/c astrocytes nor in nervous tissue, we propose an important role for CXCL1 in this experimental model of multiple sclerosis as a chemoattractant of destructive immune cells. (+info)
Tetraspanin CD151 is expressed in osteoarthritic cartilage and is involved in pericellular activation of pro-matrix metalloproteinase 7 in osteoarthritic chondrocytes.
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OBJECTIVE: The proenzyme of matrix metalloproteinase 7 (proMMP-7), which can degrade various extracellular matrix (ECM) and non-ECM molecules after being activated, is overexpressed in osteoarthritic (OA) articular cartilage, but the process of its activation in the cartilage remains unknown. The present study was undertaken to investigate the expression of tetraspanin CD151 in OA cartilage and its involvement in proMMP-7 activation. METHODS: The expression of CD151 in articular cartilage was examined by reverse transcription-polymerase chain reaction (RT-PCR), real-time PCR, immunohistochemistry, in situ hybridization, and immunoblotting. Chondrocytes were used to study the interaction between CD151 and proMMP-7, and activation of proMMP-7. RESULTS: RT-PCR revealed expression of CD151 messenger RNA in all OA cartilage samples, but in only 30% of normal control cartilage samples. Immunohistochemistry and in situ hybridization findings indicated that CD151 was coexpressed with proMMP-7 in chondrocytes, mainly in the superficial and transitional zones of OA cartilage. CD151 immunoreactivity directly correlated with the Mankin score (r = 0.757, P < 0.0001 [n = 30]) and the degree of chondrocyte cloning (r = 0.83, P < 0.0001 [n = 30]) in the cartilage samples. Complexes CD151 and proMMP-7 and their colocalization on the cell membranes were demonstrated by immunoprecipitation and double fluorescence immunostaining of the OA chondrocytes. In situ zymography indicated that chondrocytes exhibit pericellular proteolytic activity, which was abolished by treatment with MMP inhibitors, anti-MMP-7 antibody, or anti-CD151 antibody. CONCLUSION: These data demonstrate that CD151 is overexpressed in OA cartilage and suggest that CD151 plays a role in the pericellular activation of proMMP-7, leading to cartilage destruction and/or chondrocyte cloning. (+info)