sirolimus and rottlerin

Rottlerin is a pharmacological inhibitor of protein kinase C (PKC) theta, a novel PKC selectively expressed in T lymphocytes. PKC theta is known to regulate T cell receptor (TCR)/CD28 signalling pathways in T lymphocytes, but the impact of PKC theta inhibition on human T cell responses remains undefined. In this work, we describe the effects of rottlerin on the responses of CD4+ and CD8+ human T lymphocytes upon polyclonal activation. We observed a dose-dependent inhibition of CD4+ and CD8+ T cell proliferation in response to anti-CD3/anti-CD28 antibodies stimulation in the presence of rottlerin. This inhibition was associated with impaired CD25 expression and decreased interleukin (IL)-2 production in activated T cells. In contrast, rottlerin did not alter IL-2-induced T cell proliferation. Furthermore, we demonstrated that rottlerin blocked interferon (IFN) gamma, IL-10 and IL-13 mRNA expression in TCR/CD28 activated CD4+ T cells. These findings place rottlerin as a potent immunosuppressive agent for the development of novel therapies in T cell mediated immune disorders.

Topics: Acetophenones; Antigens, CD; Antigens, Differentiation, T-Lymphocyte; Benzopyrans; Blotting, Western; Calcium; CD28 Antigens; CD4-Positive T-Lymphocytes; CD8 Antigens; CD8-Positive T-Lymphocytes; Cell Proliferation; Cytokines; Dose-Response Relationship, Drug; Enzyme Inhibitors; Flow Cytometry; Humans; Interleukin-2; Interleukin-2 Receptor alpha Subunit; Intracellular Signaling Peptides and Proteins; Lectins, C-Type; Propidium; Protein Kinase C-delta; Protein Tyrosine Phosphatases; Protein Tyrosine Phosphatases, Non-Receptor; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sirolimus; Th1 Cells; Th2 Cells; Thymidine

The alpha(1)-adrenergic agonist, phenylephrine (PE), exerts hypertrophic effects in the myocardium and activates protein synthesis. Both Ca(2+)-dependent protein kinase C (PKC, PKCalpha) and Ca(2+)-independent PKC isoforms (PKCdelta and epsilon ) are detectably expressed in adult rat cardiomyocytes. Stimulation of the alpha(1)-adrenergic receptor by PE results in activation of Ca(2+)-independent PKCs, as demonstrated by translocation of the delta and epsilon isoenzymes from cytosol to membrane fractions. PE also induces activation of p70 ribosomal protein S6 kinases (S6K1 and 2) in adult cardiomyocytes. We have studied the role of Ca(2+)-independent PKCs in the regulation of S6K activity by PE. Activation of S6K1/2 by PE was blocked by the broad-spectrum PKC inhibitor bisindolylmaleimide (BIM) I, whereas Gö6976, a compound that only inhibits Ca(2+)-dependent PKCs, did not inhibit S6K activation. Rottlerin, which selectively inhibits PKCdelta, also prevented PE-induced S6K activation. The isoform-specific PKC inhibitors had similar effects on the phosphorylation of eukaryotic initiation factor 4E (eIF4E)-binding protein 1, a translation repressor that, like the S6Ks, lies downstream of the mammalian target of rapamycin (mTOR). Infection of cells with adenoviruses encoding dominant-negative PKCdelta or epsilon inhibited the activation of extracellular-signal-regulated kinase (ERK) by PE, and also inhibited the activation and/or phosphorylation of S6Ks 1 and 2. The PE-induced activation of protein synthesis was abolished by BIM I and markedly attenuated by rottlerin. Our data thus suggest that Ca(2+)-independent PKC isoforms play an important role in coupling the alpha(1)-adrenergic receptor to mTOR signalling and protein synthesis in adult cardiomyocytes.

Topics: Acetophenones; Adenoviridae; Adrenergic alpha-1 Receptor Agonists; Adrenergic alpha-Agonists; Animals; Benzopyrans; Calcium; Enzyme Activation; Enzyme Inhibitors; Genes, Dominant; Indoles; Maleimides; Mitogen-Activated Protein Kinases; Myocytes, Cardiac; Phenylephrine; Phosphorylation; Protein Isoforms; Protein Kinase C; Protein Transport; Rats; Receptors, Adrenergic, alpha-1; Ribosomal Protein S6 Kinases, 90-kDa; Signal Transduction; Sirolimus

WY-14,643, a potent hepatic peroxisome proliferator, decreased gap junctional intercellular communication when used at low and intermediate concentrations (1 nM to 10 microM) in the V79 Chinese hamster fibroblast cell line. It did not decrease intercellular communication in early passage Syrian hamster embryo fibroblasts. The mechanism of WY-14,643-induced suppression of intercellular communication was studied by preexposure of V79 cells to inhibitors of protein kinase C, mitogen-activated protein kinases, phosphatidylinositol 3-kinase, or mammalian target-of-rapamycin before addition of WY-14,643. Only protein kinase C, particularly the delta isoform, appeared involved in the inhibition of communication by WY-14,643. Also clofibrate-induced suppression of GJIC in V79 cells appeared to involve protein kinase Cdelta. Furthermore, WY-14,643 did not cause any activation of the mitogen-activated protein kinases p44/42, p38, or Jun N-terminal kinase. When WY-14,643 was used at a higher concentration (100 microM), intercellular communication was increased both in V79 and Syrian hamster embryo cells. This effect was inhibited by preexposure of V79 cells to brefeldin A. Thus, there may be a pool of connexins in the Golgi complex that could be recruited to the plasma membrane upon exposure to high concentrations of WY-14,643.

Topics: Acetophenones; Androstadienes; Animals; Benzopyrans; Brefeldin A; Butadienes; Cell Communication; Cells, Cultured; Cricetinae; Cricetulus; Enzyme Inhibitors; Flavonoids; Gap Junctions; Imidazoles; Indoles; Maleimides; Mesocricetus; Mitogen-Activated Protein Kinases; Nitriles; Peroxisome Proliferators; Protein Kinase C; Protein Synthesis Inhibitors; Pyridines; Pyrimidines; Signal Transduction; Sirolimus; Wortmannin

The specificities of 28 commercially available compounds reported to be relatively selective inhibitors of particular serine/threonine-specific protein kinases have been examined against a large panel of protein kinases. The compounds KT 5720, Rottlerin and quercetin were found to inhibit many protein kinases, sometimes much more potently than their presumed targets, and conclusions drawn from their use in cell-based experiments are likely to be erroneous. Ro 318220 and related bisindoylmaleimides, as well as H89, HA1077 and Y 27632, were more selective inhibitors, but still inhibited two or more protein kinases with similar potency. LY 294002 was found to inhibit casein kinase-2 with similar potency to phosphoinositide (phosphatidylinositol) 3-kinase. The compounds with the most impressive selectivity profiles were KN62, PD 98059, U0126, PD 184352, rapamycin, wortmannin, SB 203580 and SB 202190. U0126 and PD 184352, like PD 98059, were found to block the mitogen-activated protein kinase (MAPK) cascade in cell-based assays by preventing the activation of MAPK kinase (MKK1), and not by inhibiting MKK1 activity directly. Apart from rapamycin and PD 184352, even the most selective inhibitors affected at least one additional protein kinase. Our results demonstrate that the specificities of protein kinase inhibitors cannot be assessed simply by studying their effect on kinases that are closely related in primary structure. We propose guidelines for the use of protein kinase inhibitors in cell-based assays.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Acetophenones; Alkaloids; Amides; Animals; Benzamides; Benzophenanthridines; Benzopyrans; Butadienes; Cell Line; Enzyme Inhibitors; Flavonoids; Humans; Imidazoles; Indoles; Inhibitory Concentration 50; Isoquinolines; Lithium; Magnesium; Nitriles; Phenanthridines; Phosphorylation; Potassium Chloride; Protein Kinase Inhibitors; Protein Kinases; Pyridines; Sirolimus; Substrate Specificity; Sulfonamides