LACTATE-DEHYDROGENASE-A SUBUNIT MESSENGER-RNA STABILITY IS SYNERGISTICALLY REGULATED VIA THE PROTEIN-KINASE-A AND PROTEIN-KINASE-C SIGNAL-TRANSDUCTION PATHWAYS

We have identified and studied a posttranscriptional mechanism of lact ate dehydrogenase A (LDH) subunit gene expression at the level of mRNA stability. Using the well differentiated rat C6 glioma cell line as a model system, the effects of activators of the protein kinase A and C pathways on the half-life of LDH A mRNA were measured by two independ ent methods: 1) by the RNA synthesis inhibitor-chase method using acti nomycin D, and 2) by analysis of decay of LDH A [H-3]mRNA in [H-3]urid ine-labeled cells. By each method, the half-life of relatively short-l ived LDH A mRNA was increased 5- to 7-fold in 8- (4-chloro-phenylthio) cAMP or forskolin-treated and about 3-fold in 12-0-tetradecanoylphorb ol-13- acetate (TPA) or dioctanoylglycerol-treated cells. Forskolin ac ted synergistically with TPA to prolong LDH A mRNA half-life from 55 m in to more than 20 h. The relatively rapid basal decay rate of LDH A m RNA was also considerably slowed in the presence of the protein phosph atase inhibitor okadaic acid, suggesting a functional role for protein phosphorylation in the stabilization process. In glioma cells stably transformed with a protein kinase A catalytic subunit expression vecto r, overexpression of the catalytic subunit stabilized LDH mRNA to the degree seen in forskolin-treated cells. In cells transfected with a pr otein kinase A inhibiter-expression vector, cAMP-mediated stabilizatio n of LDH A mRNA half-life was prevented. Furthermore, both staurospori n and 3- [1-(3-dimethylaminopropyl)indol-3-yl]-3-(indol- 3-yl)-maleimi de, inhibitors of protein kinase C, prevented the TPA-induced stabiliz ation of LDH A mRNA. We conclude from the experimental data that the p rotein kinase A and C signal pathways play an active functional role i n regulating LDH A mRNA stability and act cooperatively to achieve LDH A mRNA stability regulation.