Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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C4b-binding protein protects beta-cells from islet amyloid polypeptide induced cytotoxicity.

ABSTRACT: Complement inhibitor C4b-binding protein (C4BP) is synthesized in liver and pancreas and composed of 7 identical alpha chains and one unique beta chain. We showed previously that C4BP binds islet amyloid polypeptide (IAPP) and affects fibril formation in vitro. Now we found that polymeric C4BP inhibited lysis of human erythrocytes incubated with monomeric IAPP while no erythrocyte lysis was observed after incubation with preformed IAPP fibrils. In contrast, monomeric alpha chain of C4BP had significantly reduced activity. Further, addition of monomeric IAPP to a rat insulinoma cell line (INS-1) resulted in decreased cell viability, which was restored in the presence of physiological concentrations of C4BP. Accordingly, addition of C4BP rescued the ability of INS-1 cells and isolated rat islets to respond to glucose stimulation with insulin secretion, which was impaired in the presence of IAPP alone. C4BP was internalized together with IAPP into INS-1 cells and therefore we aimed to study its effect on gene expression. Pathway analyses of mRNA expression microarray data indicated that cells exposed to C4BP and IAPP in comparison to IAPP alone increased expression of genes involved in cholesterol synthesis. Depletion of cholesterol through methyl-Î²-cyclodextrin or cholesterol oxidase abolished the protective effect of C4BP on IAPP cytotoxicity of INS-1 cells. Also, inhibition of phosphoinositide 3-kinase but not NF-ÎºB had a similar effect. Taken together, one of the mechanisms by which C4BP protects beta-cells from IAPP cytotoxicity is by enhancing cholesterol synthesis. The INS-1 cells were grown as 5 separate clones for 10 passages before plating in a 12-well plate (Nunc) at 100.000 cells per well and grown in complete RPMI 1640 medium to 70% confluency for approximately 48 h. The cells were then challenged by adding 77 Î¼M monomeric IAPP alone or together with C4BP (0.6 Î¼M). DMSO (1%) used as solvent for IAPP as well as C4BP (0.6 Î¼M) alone were used as controls. RNA was extracted after 10h incubation and analysis carried out using Rat Gene 2.0 array chip (Affymetrix).

ORGANISM(S): Rattus norvegicus

SUBMITTER: Petter Storm

PROVIDER: E-GEOD-79224 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Glioblastoma multiforme (GBM) is clinically highly aggressive as a result of evolutionary dynamics induced by cross-talk between cancer cells and a heterogeneous group of immune cells in tumor microenvironment. The brain harbors limited numbers of immune cells with few lymphocytes and macrophages; thus, innateâ€like lymphocytes, such as Î³Î´ T cells, have important roles in antitumor immunity. Here, we characterized GBMâ€infiltrating Î³Î´ T cells, which may have roles in regulating the GBM tumor microenvironment and cancer cell gene expression. V(D)J repertoires of tumorâ€infiltrating and bloodâ€circulating Î³Î´ T cells from four patients were analyzed by next-generation sequencing-based T-cell receptor (TCR) sequencing in addition to mutation and immune profiles in four GBM cases. In all tumor tissues, abundant innate and effector/memory lymphocytes were detected, accompanied by large numbers of tumorâ€associated macrophages and closely located tumorâ€infiltrating Î³Î´ T cells, which appear to have anti-tumor activity. The immune-related gene expression analysis using the TCGA database showed that the signature gene expression extent of Î³Î´ T cells were more associated with those of cytotoxic T and Th1 cells and M1 macrophages than those of Th2 cells and M2 macrophages. Although the most abundant Î³Î´ T cells were VÎ³9VÎ´2 T cells in both tumor tissues and blood, the repertoire of intratumoral VÎ³9VÎ´2 T cells was distinct from that of peripheral blood VÎ³9VÎ´2 T cells and was dominated by VÎ³9JÎ³2 sequences, not by canonical VÎ³9JÎ³P sequences that are mostly commonly found in blood Î³Î´ T cells. Collectively, unique GBMâ€specific TCR clonotypes were identified by comparing TCR repertoires of peripheral blood and intraâ€tumoral Î³Î´ T cells. These findings will be helpful for the elucidation of tumor-specific antigens and development of anticancer immunotherapies using tumor-infiltrating Î³Î´ T cells.

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C4b-binding protein protects beta-cells from islet amyloid polypeptide induced cytotoxicity.

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