rottlerin and phorbolol-myristate-acetate

Previously we have shown that a fraction of the matrix metalloproteinase-9 (MMP-9) synthesized by the macrophage cell line THP-1 was bound to a chondroitin sulphate proteoglycan (CSPG) core protein as a reduction sensitive heteromer. Several biochemical properties of the enzyme were changed when it was bound to the CSPG.. By use of affinity chromatography, zymography, and radioactive labelling, various macrophage stimulators were tested for their effect on the synthesis of the proMMP-9/CSPG heteromer and its components by THP-1 cells. Of the stimulators, only PMA largely increased the biosynthesis of the heteromer. As PMA is an activator of PKC, we determined which PKC isoenzymes were expressed by performing RT-PCR and Western Blotting. Subsequently specific inhibitors were used to investigate their involvement in the biosynthesis of the heteromer. Of the inhibitors, only Rottlerin repressed the biosynthesis of proMMP-9/CSPG and its two components. Much lower concentrations of Rottlerin were needed to reduce the amount of CSPG than what was needed to repress the synthesis of the heteromer and MMP-9. Furthermore, Rottlerin caused a minor reduction in the activation of the PKC isoenzymes δ, ε, θ and υ (PKD3) in both control and PMA exposed cells.. The biosynthesis of the proMMP-9/CSPG heteromer and proMMP-9 in THP-1 cells involves a Rottlerin-sensitive pathway that is different from the Rottlerin sensitive pathway involved in the CSPG biosynthesis. MMP-9 and CSPGs are known to be involved in various physiological and pathological processes. Formation of complexes may influence both the specificity and localization of the enzyme. Therefore, knowledge about biosynthetic pathways and factors involved in the formation of the MMP-9/CSPG heteromer may contribute to insight in the heteromers biological function as well as pointing to future targets for therapeutic agents.

Topics: Acetophenones; Benzopyrans; Cell Line; Cell Survival; Chondroitin Sulfate Proteoglycans; Dose-Response Relationship, Drug; Enzyme Activation; Gene Expression Regulation, Enzymologic; Humans; Isoenzymes; Macrophages; Matrix Metalloproteinase 9; Protein Kinase C; Protein Multimerization; Protein Structure, Quaternary; RNA, Messenger; Signal Transduction; Tetradecanoylphorbol Acetate; Time Factors

1. Staurosporine is a broad-specificity kinase inhibitor, which has acted as lead compound for the development of some novel cytotoxic compounds for treatment of cancer. This study investigates the unexpected observation that staurosporine can also induce homotypic cellular aggregation. 2. In this study, staurosporine is shown to activate rapid homotypic aggregation of U937 cells, at concentrations below those required to induce cell death. This activity is a particular feature of staurosporine, and is not shared by a number of other kinase inhibitors. The proaggregating activity of staurosporine is inhibited by deoxyglucose, cytochalasin B and colchicine. Staurosporine-induced aggregation can be distinguished from that induced by the phorbol 12-myristate 13-acetate by faster kinetics and insensitivity to cycloheximide. Staurosporine induces translocation of conventional and novel, but not atypical isoforms of protein kinase C (PKC). Aggregation induced by staurosporine is inhibited by a number of inhibitors of PKC isoforms, and by inhibitors of protein tyrosine kinases. Staurosporine also induces rapid phosphorylation of ERK and p38, and inhibitors of both these enzymes block aggregation. 3. Staurosporine induces dysregulated activation of multiple kinase signaling pathways in U937 cells, and the combined activity of several of these pathways is essential for the induction of aggregation.

Topics: Acetophenones; Adenosine Triphosphate; Benzopyrans; Calcium-Calmodulin-Dependent Protein Kinases; Carbazoles; CD18 Antigens; Cell Aggregation; Cycloheximide; Cytoskeleton; Dose-Response Relationship, Drug; Enzyme Activation; Enzyme Inhibitors; Flavonoids; Genistein; Humans; Imidazoles; Indoles; Integrin beta1; Isoenzymes; Maleimides; Mitogen-Activated Protein Kinases; Naphthalenes; Phosphorylation; Phosphotransferases; Protein Kinase C; Protein-Tyrosine Kinases; Pyridines; Staurosporine; Temperature; Tetradecanoylphorbol Acetate; Time Factors; Tyrosine; U937 Cells