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 p hosphodiesterases (PDEs) is of crucial importance for the regulation o f cAMP levels, However, PDE isoenzymes in human keratinocytes have not been characterized previously, In the present study, the PDE isoenzym e activity profile of the human keratinocyte cell line HaCaT was inves tigated by PDE activity measurements, In addition, the cAMP-mediated r egulation of PDE activities was examined, The isoenzymes PDE IV and PD E V activities were identified in HaCaT cell homogenates by activity m easurements 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 affec ted, 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 cyclo heximide indicated that de novo mRNA and protein synthesis were at lea st partly involved in PDE IV upregulation, Functionally, the enhanced PDE IV activity was reflected by an impaired cAMP response to salbutam ol, This hyporesponsiveness toward the beta(2)-adrenoceptor agonists w as partly reversed by rolipram. This study describes a cAMP-dependent longterm up-regulation of PDE IV in HaCaT cells, which is at least par tly reflected by a simultaneous reduced cAMP response to a beta-agonis t.