PROTEIN-KINASE-A REGULATION OF CAMP-PHOSPHODIESTERASE EXPRESSION IN RAT SKELETAL MYOBLASTS

We have been studying cAMP signaling in L6 myoblasts because of its po tential role in regulating the differentiation of these cells into mul tinucleate myotubes. Previous studies have shown that treatment of L6 myoblasts with cAMP analogs causes an increase in cAMP phosphodiestera se activity. To assess the role of protein kinase A in this cAMP-media ted increase in cAMP phosphodiesterase activity, L6 myoblasts were tra nsfected with a plasmid containing the cDNA for a mutant regulatory su bunit of protein kinase A, which functions as a dominant negative inhi bitor of this enzyme. The cDNA was under control of the metallothionei n promoter in the construct. Induction of the mutant regulatory subuni t with Zn2+ decreased cAMP-dependent protein kinase activity by 90%. Z n2+ treatment was also able to completely block the cAMP-mediated incr ease in phosphodiesterase activity, showing that this effect is mediat ed by protein kinase A. The activity of the cAMP-induced phosphodieste rase was inhibited by low concentrations of RO 20-1724, showing that i t was a member of the type IV low K-m cAMP phosphodiesterase family of enzymes. We used the polymerase chain reaction and consensus primers designed to amplify phosphodiesterase sequences to show that L6 myobla sts also contain mRNA for a type IV low K-m cAMP phosphodiesterase des ignated PDES3.1. The levels of this mRNA were increased greatly by tre atment with dibutyryl cAMP or forskolin in L6 myoblasts and also in di fferentiated L6 myotubes. Run off transcription assays showed that thi s increase in PDE mRNA was regulated, at least in part, by an increase in the rate of transcription of the PDES gene. The induction of PDES message by cAMP was blocked when the L6 transfectants were treated wit h Zn2+ to induce protein kinase A inhibition. Therefore, some of the c AMP-mediated increase in phosphodiesterase activity seen in L6 myoblas ts is due to a protein kinase A-mediated increase in PDE3 mRNA. This p athway may serve as a feedback mechanism to modulate the inhibitory ef fects of cAMP on myogenesis.