Identification of phosphodiesterase IV activity and its cyclic adenosine monophosphate-dependent up-regulation in a human keratinocyte cell line (HaCaT).
Cellular activity of cyclic adenosine monophosphate (cAMP)-degrading phosphodiesterases (PDEs) is of crucial importance for the regulation of cAMP levels. However, PDE isoenzymes in human keratinocytes have not been characterized previously. In the present study, the PDE isoenzyme activity profile of the human keratinocyte cell line HaCaT was investigated by PDE activity measurements. In addition, the cAMP-mediated regulation of PDE activities was examined. The isoenzymes PDE IV and PDE V activities were identified in HaCaT cell homogenates by activity measurements and were found to be preferentially located in the soluble fraction. Long-term exposure of HaCaT cells to cAMP-elevating agents (e.g., rolipram, salbutamol, forskolin) triggered a maximum threefold up-regulation of PDE IV activity, whereas PDE V activity was not affected. The PDE IV inhibitor rolipram synergistically amplified PDE IV up-regulation by beta 2-receptor agonists. Experiments applying protein kinase A activators and inhibitors as well as actinomycin D and cycloheximide indicated that de novo mRNA and protein synthesis were at least partly involved in PDE IV up-regulation. Functionally, the enhanced PDE IV activity was reflected by an impaired cAMP response to salbutamol. This hyporesponsiveness toward the beta 2-adrenoceptor agonists was partly reversed by rolipram. This study describes a cAMP-dependent long-term up-regulation of PDE IV in HaCaT cells, which is at least partly reflected by a simultaneous reduced cAMP response to a beta-agonist.
Tenor
H
H
Faculty of Biology, University of Konstanz, Germany.
Hatzelmann
A
A
Wendel
A
A
Schudt
C
C
D015105:3',5'-Cyclic-AMP Phosphodiesterases; D000420:Albuterol / Q000494:pharmacology; D002460:Cell Line; D000242:Cyclic AMP / Q000502:physiology*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000502:physiology; D054703:Cyclic Nucleotide Phosphodiesterases, Type 4; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism; D015603:Keratinocytes / Q000201:enzymology*; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism*; D011760:Pyrrolidinones / Q000494:pharmacology; D020889:Rolipram; D015854:Up-Regulation
D007527:Isoenzymes; D011760:Pyrrolidinones; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D010727:Phosphoric Diester Hydrolases; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D054703:Cyclic Nucleotide Phosphodiesterases, Type 4; D020889:Rolipram; D000420:Albuterol