ag-490 and manumycin

Tobacco products and nicotine alter the cell cycle and lead to squamatization of oral keratinocytes (KCs) and squamous cell carcinoma. Activation of nicotinic acetylcholine receptors (nAChRs) elicits Ca(2+) influx that varies in magnitude between different nAChR subtypes. Normal differentiation of KCs is associated with sequential expression of the nAChR subtypes with increasing Ca(2+) permeability, such as alpha5-containing alpha3 nAChR and alpha7 nAChR. Exposure to environmental tobacco smoke (ETS) or an equivalent concentration of nicotine accelerated by severalfold the alpha5 and alpha7 expression in KCs, which could be abolished by mecamylamine and alpha-bungarotoxin with different efficacies, suggesting the following sequence of autoregulation of the expression of nAChR subtypes: alpha3(beta2/beta4) > alpha3(beta2/beta4)alpha5 > alpha7 > alpha7. This conjecture was corroborated by results of quantitative assays of subunit mRNA and protein levels, using nAChR-specific pharmacologic antagonists and small interfering RNAs. The genomic effects of ETS and nicotine involved the transcription factor GATA-2 that showed a multifold increase in quantity and activity in exposed KCs. Using protein kinase inhibitors and dominant negative and constitutively active constructs, we characterized the principal signaling cascades mediating a switch in the nAChR subtype. Cumulative results indicated that the alpha3(beta2/beta4) to alpha3(beta2/beta4)alpha5 nAChR transition predominantly involved protein kinase C, alpha3(beta2/beta4)alpha5 to alpha7 nAChR transition-Ca(2+)/calmodulin-dependent protein kinase II and p38 MAPK, and alpha7 self-up-regulation-the p38 MAPK/Akt pathway, and JAK-2. These results provide a mechanistic insight into the genomic effects of ETS and nicotine on KCs and characterize signaling pathways mediating autoregulation of stepwise overexpression of nAChR subtypes with increasing Ca(2+) permeability in exposed cells. These observations have salient clinical implications, because a switch in the nAChR subunit composition can bring about a corresponding switch in receptor function, leading to profound pathobiologic effects observed in KCs exposed to tobacco products.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; alpha7 Nicotinic Acetylcholine Receptor; Bungarotoxins; Butadienes; Carbazoles; Conotoxins; Egtazic Acid; GATA2 Transcription Factor; Humans; Imidazoles; Indoles; Keratinocytes; Nerve Tissue Proteins; Nicotine; Nitriles; Phenols; Polyenes; Polyunsaturated Alkamides; Pyridines; Receptors, Nicotinic; RNA, Small Interfering; Signal Transduction; Smoking; Tobacco Smoke Pollution; Tyrphostins; Up-Regulation

Malignant transformation of cells is frequently associated with an augmented production of hyaluronan and the subsequent formation of a hyaluronan-matrix. In v-Src-transformed cells, hyaluronan directly activate cell motility in a tumor-specific manner. Despite its importance, the mechanism by which v-Src activates hyaluronan production remains unclear. Here we report that multiple signaling pathways are required for the augmented production of hyaluronan. Either the expression of a dominant negative Ras or the treatment of cells with manumycin A, a Ras farnesyltransferase inhibitor, was able to suppress hyaluronan production. In contrast, expression of MEK1EE, a constitutive form of MEK1, activated both hyaluronan synthase expression and hyaluronan production. AG-490, a Jak-2 inhibitor, or LY294002, a PI3K inhibitor, similarly suppressed the augmented production of hyarulonan. Taken together, our results suggest the involvement of multiple signaling pathways, including Ras-dependent and independent ones, in augmented hyaluronan production by v-Src.

Topics: Animals; Cell Line; Chromones; Dimethyl Sulfoxide; Enzyme Inhibitors; Flavonoids; Gene Expression; Glucuronosyltransferase; Hyaluronan Synthases; Hyaluronic Acid; Immunoblotting; Isoenzymes; Janus Kinase 2; MAP Kinase Kinase 1; Morpholines; Mutation; Oncogene Protein pp60(v-src); Polyenes; Polyunsaturated Alkamides; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; Time Factors; Tyrphostins