h-89 and Fibrosarcoma

ATP-binding cassette transporter A1 (ABCA1) mediates transport of cellular cholesterol and phospholipids to high density lipoprotein (HDL) apolipoproteins, such as apoA-I. ABCA1 mutations can cause a severe HDL deficiency and atherosclerosis. Here we show that the protein-tyrosine kinase (TK) Janus kinase 2 (JAK2) modulates the apolipoprotein interactions with ABCA1 required for removing cellular lipids. The protein kinase A (PKA) inhibitor H89, the TK inhibitor genistein, and the JAK2 inhibitor AG490 suppressed apoA-I-mediated cholesterol and phospholipid efflux from ABCA1-expressing cells without altering the membrane ABCA1 content. Whereas PKA inhibition had no effect on apoA-I binding to cells or to ABCA1, TK and JAK2 inhibition greatly reduced these activities. Conversely, PKA but not JAK2 inhibition significantly reduced the intrinsic cholesterol translocase activity of ABCA1. Mutant cells lacking JAK2 had a severely impaired apoA-I-mediated cholesterol and phospholipid efflux and apoA-I binding despite normal ABCA1 protein levels and near normal cholesterol translocase activity. Thus, although PKA modulates ABCA1 lipid transport activity, JAK2 appears to selectively modulate apolipoprotein interactions with ABCA1. TK-mediated phosphorylation of ABCA1 was undetectable, implicating the involvement of another JAK2-targeted protein. Acute incubation of ABCA1-expressing cells with apoA-I had no effect on ABCA1 phosphorylation but stimulated JAK2 autophosphorylation. These results suggest that the interaction of apolipoproteins with ABCA1-expressing cells activates JAK2, which in turn activates a process that enhances apolipoprotein interactions with ABCA1 and lipid removal from cells.

Topics: Animals; Apolipoprotein A-I; Apolipoproteins; ATP Binding Cassette Transporter 1; ATP-Binding Cassette Transporters; Cell Line; Cholesterol; Cricetinae; Enzyme Inhibitors; Fibrosarcoma; Genistein; Humans; Isoquinolines; Janus Kinase 2; Kidney; Mutation; Phospholipids; Phosphorylation; Protein-Tyrosine Kinases; Proto-Oncogene Proteins; Recombinant Proteins; RNA, Messenger; Sulfonamides; Transfection; Tumor Cells, Cultured; Tyrosine; Tyrphostins

We evaluated the role of MAPKs on apoptosis induced by butyrate in cells derived from a human fibrosarcoma (2C4). Culture of 2C4 cells in 5% of fetal bovine serum (FBS) induced ERK1/2 and CREB phosphorylation and delayed apoptosis induced by butyrate. Butyrate inhibited phosphorylation of ERK1/2 and CREB. Furthermore, the use of specific inhibitors PD98059 (MEK) and H89 (PKA), which block ERK1/2 and CREB phosphorylation, accelerated butyrate induced cell death in 2C4 cells. The butyrate effect was shown to be dependent on caspase activation, once caspase inhibitors restored phosphorylation of ERK1/2 and CREB in 2C4 cells. However, the proteolytic effect of caspases was not directly on ERK1/2 and CREB proteins. In conclusion, butyrate induced apoptosis in 2C4 cells is regulated by the levels of ERK1/2 and CREB phosphorylation in a caspase dependent mechanism.

Topics: Antineoplastic Agents; Apoptosis; Butyrates; Caspases; Cyclic AMP Response Element-Binding Protein; Enzyme Inhibitors; Fibrosarcoma; Flavonoids; Humans; Isoquinolines; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Phosphorylation; Signal Transduction; Sulfonamides; Tumor Cells, Cultured