REQUIREMENT OF MAP KINASE FOR DIFFERENTIATION OF FIBROBLASTS TO ADIPOCYTES, FOR INSULIN ACTIVATION OF P90 S6 KINASE AND FOR INSULIN OR SERUM STIMULATION OF DNA-SYNTHESIS

A phosphorothioate-oligonucleotide-based antisense strategy for deplet ing MAP kinase was developed, The 17mer antisense probe, EAS 1, caused a potent and concentration-dependent decrease in the steady state exp ression of p42 and p44 MAP kinase in 3T3 L1 fibroblasts and adipocytes with submicromolar concentrations effective, Antisense EAS 1 elicited a dose-dependent inhibition of insulin- and serum-stimulated DNA synt hesis, Elimination of p42 MAP kinase by >95% and p44 MAP kinase to lev els undetected blocked the ability of serum in 3T3 L1 fibroblasts and insulin in 3T3 L1 adipocytes to stimulate DNA synthesis by 87-95%, The differentiation of 3T3 L1 fibroblasts into adipocytes was prevented b y 1 mu M antisense EAS 1. The corresponding sense, scrambled or sense plus antisense EAS 1 phosphorothioate oligonucleotides did not deplete the p42 or p44 MAP kinase from either cell type, did not inhibit stim ulation of DNA synthesis and did not interfere with differentiation. T wo kinases on different MAP kinase activation pathways were not deplet ed by antisense EAS 1 whereas the ability of insulin to activate p90 S 6 kinase was >90% eliminated in 3T3 L1 adipocytes by 4.5 mu M antisens e EAS 1, In conclusion these results show that MAP kinase is required for insulin and serum stimulation of DNA synthesis, for insulin stimul ation of p90 S6 kinase activity and for differentiation of 3T3 L1 cell s, Moreover, the development of the antisense probe EAS 1 against a ta rget sequence of p42 MAP kinase that is conserved in p44 MAP kinase an d across a range of species provides a molecular tool of general appli cability for further dissecting the precise targets and roles of MAP k inase.