ucn-1028-c and arginyl-glycyl-aspartic-acid

Interleukin 1beta (IL-1beta) is a multifunctional polypeptide considered a key cytokine during inflammation. Fibronectin (FN), a matrix glycoprotein highly expressed in injured tissues, can induce expression of IL-1beta in human blood monocytic cells. Herein, we explore the intracellular signals and transcriptional mechanisms responsible for IL-1beta induction by FN using human promonocytic U937 cells transfected with the human IL-1beta promoter connected to a reporter gene. Exposure of transfected U937s to FN resulted in increased expression of the full-length IL-1beta promoter. This effect, mediated via the alpha5beta1 integrin, was associated with activation of mitogen-activated protein kinases (MAPKs) and was abolished by pre-treatment of cells with Calphostin C, a specific inhibitor of protein kinase C (PKC) activation. Deletion analysis and co-transfection studies using consensus activator protein 1 (AP-1) oligonucleotides suggested that an AP-1 site present in the 5' end of the IL-1beta promoter was involved in the FN-induced response. Finally, electrophoretic mobility shift assays showed that FN induced binding of AP-1, but not NF-kappaB. Together, these experiments demonstrate that FN binding to the alpha5beta1 integrin activates MAPK-dependent signal pathways, and results in the transcription of the IL-1beta promoter in U937 cells by activating PKC and inducing AP-1.

Topics: Blotting, Northern; Blotting, Western; Cell Adhesion; Chloramphenicol O-Acetyltransferase; Dose-Response Relationship, Drug; Electroporation; Enzyme Activation; Enzyme Inhibitors; Fibronectins; Gene Deletion; Gene Expression Regulation; Genes, Reporter; Humans; Interleukin-1; Lipopolysaccharides; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Naphthalenes; NF-kappa B; Oligopeptides; Precipitin Tests; Promoter Regions, Genetic; Protein Kinase C; Receptors, Fibronectin; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; Tetradecanoylphorbol Acetate; Transcription Factor AP-1; Transcription, Genetic; Transfection; U937 Cells

Phorbol 12-myristate 13-acetate (PMA) is a tumour promotor that acts as a potent protein kinase C (PKC) activator that has significant effects on tumour cell attachment and spreading. We tested whether these effects of PMA may be observed in human melanoma cells, and whether a specific response to extracellular matrix proteins may be mediated by shifts in the expression of beta 1 integrins. We used cell attachment assays, video time lapse cell spreading assays, flow cytometry, function blocking monoclonal antibodies (MAbs) and PKC inhibitor Calphostin C to address these questions. We established that PMA induces a rapid and temporary enhancement of cell attachment and spreading which was not accompanied by a significant change in the expression of beta 1 integrins. Spreading of melanoma cells that were not stimulated with PMA could be significantly blocked with a function blocking MAb (clone P4C10) against the common beta 1 integrin subunit. The spreading and attachment of the PMA treated cells was also significantly reduced, but less so, after MAb treatment. The PMA enhanced cell attachment and spreading could be effectively blocked by RGD sequences and PKC inhibitor. Taken together, our data indicate that PMA induces a rapid and temporary ECM-dependent enhancement of melanoma cell attachment and spreading, and that the response to ECM components appears not to be due to significant shifts in beta 1 integrin expression, but rather to activation of beta 1 integrins.

Topics: Amino Acid Sequence; Cell Adhesion; Extracellular Matrix; Extracellular Matrix Proteins; Fibronectins; Humans; Integrins; Melanoma; Molecular Sequence Data; Naphthalenes; Oligopeptides; Polycyclic Compounds; Protein Kinase C; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured