Ca2+ potentiates cAMP-dependent expression of urokinase-type plasminogen activator gene through a calmodulin- and protein kinase C-independent mechanism.
In the porcine renal epithelial cell line, LLC-PK1, activation of the cAMP-dependent signal transduction pathway induces the urokinase-type plasminogen activator (uPA) gene. We show here that the cAMP response is enhanced when the intracellular calcium concentration is increased. When LLC-PK1 cells were treated with the calcium ionophore ionomycin alone, there was no uPA mRNA accumulation. However, in the presence of ionomycin the dose-response of 8-bromo-cAMP (Br-cAMP) with respect to uPA mRNA accumulation was shifted toward the lower concentrations of Br-cAMP. A Northern blot analysis after the inhibition of RNA synthesis and nuclear run-on assays showed that the synergistic effect of Ca2+ could be attributed to increases in uPA gene transcription and mRNA stability. In the presence of cycloheximide, a protein synthesis inhibitor, uPA mRNA was stabilized, but the effect of ionomycin on Br-cAMP-induced mRNA accumulation was still maintained. The result suggests that the Ca2+, at least on transcription, does not require new protein synthesis. Ionomycin treatment did not modify the activity of the cAMP-dependent protein kinase, suggesting that Ca2+ either affects a step in the pathway between the kinase and the uPA gene, or acts independently of the cAMP-dependent protein kinase pathway. The effect of ionomycin was not suppressed by protein kinase C down-regulation nor by inhibitors of calmodulin. Synergism was also observed when Br-cAMP was replaced with calcitonin, a peptide hormone which is coupled to adenylate cyclase, and when ionomycin was replaced with another ionophore A23187, suggesting that the synergism is due to an interaction between cAMP-dependent and Ca2(+)-dependent signal transduction pathways.
Friedrich Miescher-Institut, Basel, Switzerland.
A
A
Ziegler
J
J
Hagmann
B
B
Kiefer
Y
Y
Nagamine
D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000494:pharmacology; D000818:Animals; D015152:Blotting, Northern; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology*; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002460:Cell Line; D003001:Cloning, Molecular; D000242:Cyclic AMP / Q000502:physiology*; D004792:Enzyme Precursors / Q000235:genetics; D015870:Gene Expression / Q000187:drug effects*; D015759:Ionomycin / Q000494:pharmacology; D007700:Kinetics; D009693:Nucleic Acid Hybridization; D010957:Plasmids; D010960:Plasminogen Activators / Q000235:genetics*; D011493:Protein Kinase C / Q000378:metabolism; D011494:Protein Kinases / Q000378:metabolism; D012313:RNA / Q000235:genetics / Q000302:isolation & purification; D012333:RNA, Messenger / Q000187:drug effects / Q000235:genetics; D013552:Swine; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014568:Urokinase-Type Plasminogen Activator / Q000235:genetics*
D004792:Enzyme Precursors; D012333:RNA, Messenger; D015124:8-Bromo Cyclic Adenosine Monophosphate; D015759:Ionomycin; D012313:RNA; D000242:Cyclic AMP; D011494:Protein Kinases; D011493:Protein Kinase C; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D010960:Plasminogen Activators; D014568:Urokinase-Type Plasminogen Activator; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium