rottlerin and bryostatin-1

Mucin hypersecretion is a major pathological feature of many respiratory diseases, yet cellular mechanisms regulating secretion of mucin have not been fully elucidated. Previously, we reported that mucin hypersecretion induced by human neutrophil elastase involves activation of protein kinase C (PKC), specifically the delta-isoform (PKC delta). Here, we further investigated the role of PKC delta in mucin hypersecretion using both primary human bronchial epithelial cells and the human bronchial epithelial 1 cell line as in vitro model systems. Phorbol-12-myristate-13-acetate (PMA)-induced mucin hypersecretion was significantly attenuated by rottlerin, a PKC delta-selective inhibitor. Rottlerin also reduced PMA- or human neutrophil elastase-induced phosphorylation of myristoylated alanine-rich C kinase substrate (MARCKS) protein in these cells. Both secretion and MARCKS phosphorylation were significantly enhanced by the PKC delta activator bryostatin 1. A dominant-negative PKC delta construct (pEGFP-N1/PKC delta K376R) transfected into human bronchial epithelial 1 cells significantly attenuated both PMA-induced mucin secretion and phosphorylation of MARCKS, whereas transfection of a wild-type construct increased PKC delta and enhanced mucin secretion and MARCKS phosphorylation. Similar transfections of a dominant-negative or wild-type PKC epsilon construct did not affect either mucin secretion or MARCKS phosphorylation. The results suggest that PKC delta plays an important role in mucin secretion by airway epithelium via regulation of MARCKS phosphorylation.

Topics: Acetophenones; Benzopyrans; Bronchi; Bryostatins; Cell Line; Enzyme Activation; Epithelial Cells; Humans; Intracellular Signaling Peptides and Proteins; Membrane Proteins; Mucins; Myristoylated Alanine-Rich C Kinase Substrate; Phosphorylation; Protein Kinase C-delta; Protein Kinase Inhibitors; Tetradecanoylphorbol Acetate

The cAMP-response element-binding protein (CREB) is activated by phosphorylation on Ser-133 and plays a key role in the proliferative and survival responses of mature B cells to B cell receptor (BCR) signaling. The signal link between the BCR and CREB activation depends on a phorbol ester (phorbol 12-myristate 13-acetate)-sensitive protein kinase C (PKC) activity and not protein kinase A or calmodulin kinase; however, the identity and role of the PKC(s) activity has not been elucidated. We found the novel PKCdelta (nPKCdelta) activator bistratene A is sufficient to induce CREB phosphorylation in murine splenic B cells. The pharmacological inhibitor Gö6976, which targets conventional PKCs and PKCmu, has no effect on CREB phosphorylation, whereas the nPKCdelta inhibitor rottlerin blocks CREB phosphorylation following BCR cross-linking. Bryostatin 1 selectively prevents nPKCdelta depletion by phorbol 12-myristate 13-acetate when coapplied, coincident with protection of BCR-induced CREB phosphorylation. Ectopic expression of a kinase-inactive nPKCdelta blocks BCR-induced CREB phosphorylation in A20 B cells. In addition, BCR-induced CREB phosphorylation is significantly diminished in nPKCdelta-deficient splenic B cells in comparison with wild type mice. Consistent with the essential role for Bruton's tyrosine kinase and phospholipase Cgamma2 in mediating PKC activation, Bruton's tyrosine kinase- and phospholipase Cgamma2-deficient B cells display defective CREB phosphorylation by the BCR. We also found that p90 RSK directly phosphorylates CREB on Ser-133 following BCR cross-linking and is positioned downstream of nPKCdelta. Taken together, these results suggest a model in which BCR engagement leads to the phosphorylation of CREB via a signaling pathway that requires nPKCdelta and p90 RSK in mature B cells.

Topics: Acetamides; Acetophenones; Animals; B-Lymphocytes; Benzopyrans; Binding Sites; Blotting, Western; Bryostatins; Calcium-Calmodulin-Dependent Protein Kinases; Carbazoles; Cell Division; Cross-Linking Reagents; Cyclic AMP Response Element-Binding Protein; Cyclic AMP-Dependent Protein Kinases; Dose-Response Relationship, Drug; Enzyme Inhibitors; Indoles; Lactones; Macrolides; Mice; Mice, Inbred BALB C; Mice, Inbred CBA; Mitogens; Phosphorylation; Promoter Regions, Genetic; Protein Isoforms; Protein Kinase C; Protein Kinase C-delta; Protein Structure, Tertiary; Pyrans; Receptors, Antigen, B-Cell; Ribosomal Protein S6 Kinases, 90-kDa; Serine; Signal Transduction; Spiro Compounds; Tetradecanoylphorbol Acetate; Time Factors

Plasma membrane cholesterol both regulates and is regulated by effector proteins in the endoplasmic reticulum (ER) through a feedback system that is poorly understood. We now show that ER cholesterol varies over a fivefold range in response to experimental agents that act upon protein kinase C (PKC). Agents that activate Ca(2+)-dependent PKC [phorbol-12-myristate-13-acetate (PMA) and bryostatin 1] increased the level of ER cholesterol; inhibitors such as staurosporine and calphostin C decreased it. Rottlerin, a selective inhibitor of the PKC-delta isoform, also increased ER cholesterol. The esterification of plasma membrane cholesterol was altered by protein kinase C-directed agents in a corresponding fashion. Furthermore, the regulatory effect of plasma membrane cholesterol on the esterification of ER cholesterol was blocked by PKC-directed agents. These findings suggest that multiple protein kinase C isoforms participate in the regulation of ER cholesterol and therefore in cholesterol homeostasis.

Topics: Acetophenones; Benzopyrans; Bryostatins; Calcium; Cell Membrane; Cells, Cultured; Cholesterol; Dose-Response Relationship, Drug; Endoplasmic Reticulum; Enzyme Inhibitors; Fibroblasts; Humans; Lactones; Macrolides; Mitogens; Naphthalenes; Protein Isoforms; Protein Kinase C; Staurosporine; Tetradecanoylphorbol Acetate

There is evidence that protein kinase C delta (PKCdelta) is a tumor suppressor, although its physiological role has not been elucidated so far. Since important anti-proliferative signals are mediated by cell-cell contacts we studied whether PKCdelta is involved in contact-dependent inhibition of growth in human (FH109) and murine (NIH3T3) fibroblasts. Cell-cell contacts were imitated by the addition of glutardialdehyde-fixed cells to sparsely seeded fibroblasts. Downregulation of the PKC isoforms alpha, delta, epsilon, and mu after prolonged treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA, 0.1 microM) resulted in a significant release from contact-inhibition in FH109 cells. Bryostatin 1 selectively prevented TPA-induced PKCdelta-downregulation and reversed TPA-induced release from contact-inhibition arguing for a role of PKCdelta in contact-inhibition. In accordance, the PKCdelta specific inhibitor Rottlerin (1 microM) totally abolished contact-inhibition. Interestingly, immunofluorescence revealed a rapid translocation of PKCdelta to the nucleus when cultures reached confluence with a peak in early-mid G1 phase. Nuclear translocation of PKCdelta in response to cell-cell contacts could also be demonstrated after subcellular fractionation by Western blotting and by measuring PKCdelta-activity after immunoprecipitation. Transient transfection of NIH3T3 cells with a dominant negative mutant of PKCdelta induced a transformed phenotype. We conclude that PKCdelta is involved in contact-dependent inhibition of growth.

Topics: 3T3 Cells; Acetophenones; Active Transport, Cell Nucleus; Animals; Benzopyrans; Bryostatins; Cell Cycle; Cell Division; Chemotaxis; Down-Regulation; Enzyme Inhibitors; Fibroblasts; Fixatives; Glutaral; Humans; Isoenzymes; Lactones; Macrolides; Mice; Mitogens; Protein Binding; Protein Isoforms; Protein Kinase C; Protein Kinase C-delta; Tetradecanoylphorbol Acetate; Time Factors

Expression of certain mammalian protein kinase C (PKC) isoforms inhibits the proliferation of Schizosaccharomyces pombe (Goode et al., Mol. Biol. Cell 5 (1994) 907-920). We have taken advantage of this fact to determine the in vivo isoform preference of a number of PKC inhibitors, using a microtitre plate assay which allows rapid screening. This in vivo model has revealed previously unreported preferences; calphostin C is a more efficient inhibitor of the novel PKCS than chelerythrine chloride whereas the efficiencies are reversed for inhibition of the classical PKCgamma. We have also shown that the anti-leukaemic agent bryostatin 1 inhibits or activates in vivo in an isoform-specific manner.

Topics: Acetophenones; Alkaloids; Benzophenanthridines; Benzopyrans; Bryostatins; Carbazoles; Enzyme Activation; Enzyme Inhibitors; Indoles; Isoenzymes; Lactones; Macrolides; Maleimides; Naphthalenes; Phenanthridines; Protein Kinase C; Protein Kinase C-delta; Recombinant Proteins; Schizosaccharomyces; Sphingosine; Tetradecanoylphorbol Acetate; Transformation, Genetic