Induction of Phosphodiesterases 3B, 4A4, 4D1, 4D2, and 4D3 in Jurkat T-cells and in Human Peripheral Blood T-lymphocytes by 8-Bromo-cAMP and Gs-coupled Receptor Agonists

Abstract

In this study, a potential mechanism of ␤ 2 -adrenoreceptor desensitization has been explored that is based upon the enhanced degradation of cAMP by phosphodiesterase (PDE). Pretreatment of Jurkat T-cells with 8-bromo cAMP (8-Br-cAMP) or prostaglandin E 2 increased PDE3 and PDE4 activity in an actinomycin Dand cycloheximide-sensitive manner. This effect was associated with increased expression of HSPDE3B, HSPDE4A4, HSPDE4D1, HSPDE4D2, and HSPDE4D3 mRNA transcripts. Western analysis reproducibly labeled a band of immunoreactivity in vehicle-treated cells that corresponded to HSPDE4A4 (125 kDa). Although the intensity of this band was unchanged in cells treated with 8-Br-cAMP, additional 68 -72-kDa proteins (HSPDE4D2, HSPDE4D1) were labeled that were not detected after vehicle. Similar results were obtained with T-lymphocytes exposed to 8-Br-cAMP and fenoterol. However, in those experiments HSPDE4A4 and HSPDE4D1 appeared to be equally expressed in vehicleand treated cells, whereas HSPDE4D2 (72 kDa) was detected only after 8-Br-cAMP. The up-regulation of PDE activity in Jurkat T-cells abolished the ability of isoproterenol to elevate cAMP, which was partially reversed by the non-selective PDE inhibitor, 3-isobutyl-1-methylxanthine, and by the PDE3 and PDE4 inhibitors, Org 9935 and rolipram, respectively. Collectively, these data suggest that chronic treatment of T-cells with cAMPelevating agents compromises ␤ 2 -adrenoreceptor-mediated cAMP accumulation by increasing the expression of HSPDE3B and HSPDE4D gene products. Cyclic nucleotide phosphodiesterases (PDEs)1 are a heterogeneous group of immunologically distinct enzymes whose sole function is to metabolize the second messenger purine nucleotides, cAMP and cGMP, to their biologically inactive nucleotide 5Ј-monophosphates. Currently, PDEs are categorized in to seven broad families (see Ref. 1 for nomenclature) that are distinguished by a number of criteria including substrate specificity, kinetic properties, sensitivity to allosteric modulators and synthetic inhibitors, and primary amino acid sequence (2, 3). In many cases, these families comprise multiple subtypes, which suggests that the degradation of cAMP and cGMP is a highly complex and tightly regulated process. Over the last decade, the possible applications of PDE inhibitors to the treatment of an array of inflammatory diseases including asthma and atopic dermatitis has been realized. In particular, the cAMP-specific PDE, or PDE4, is viewed by the pharmaceutical industry as a viable molecular locus amenable to therapeutic intervention with selective inhibitors. The selection of this enzyme family as a possible drug target comes from the knowledge that PDE4 is the predominant, if not exclusive, regulator of cAMP homeostasis in essentially all pro-inflammatory and immune cells. Moreover, PDE4 inhibitors suppress many functional indices of cell activation that are considered pro-inflammatory (4 -7).Currently, four genes (for PDE4A, PDE4B, PDE4C, and PDE4D) have been identified in rat (8 -11), in mouse (12), ...

Pivotal role of cyclic nucleoside phosphodiesterase 4 in Tat-mediated CD4+T cell hyperactivation and HIV type 1 replication

“…In fact, other molecules are likely involved in the transmission of the signal. One possibility is that the cAMP͞phosphokinase A pathway was responsible for PDE4 activation, as previously observed in activated T cells (31). However, we found that the phosphokinase A inhibitor (Rp-8-Br-cyclic AMPS) was unable to reduce IL-2 production in cells seeded on plates coated with anti-CD3͞CD28 mAbs Ϯ Tat, suggesting that in our cellular system PDE4 was not activated by the cAMP͞phosphokinase A pathway.…”

“…Five distinct functional domains have been characterized in the Tat protein: N-terminal (amino acids 1-21), cysteine-rich (amino acids [22][23][24][25][26][27][28][29][30][31][32][33][34][35][36][37], core (amino acids 38-48), basic (amino acids 49-57), and C-terminal (amino acids 58-86͞ 101; ref. 1).…”

The human near‐term myometrial β3‐adrenoceptor but not the β2‐adrenoceptor is resistant to desensitisation after sustained agonist stimulation

“…This mechanism has been well documented in several systems including S49 lymphoma cells, platelets, hepatocytes, adipocytes and Sertoli cells (for review, Giembycz, 1996;Perry et al, 2002). It has been demonstrated, in different human blood cells, that treatment with either b 2 -AR agonists (Manning et al, 1996;Seybold et al, 1998;Ortiz et al, 2000) or with a cAMP structural analogue, 8-Bromo-cAMP, leads to an upregulation of PDE4 enzymes. More recently, new evidences for a role of cAMP on PDE4 promoters have been defined (Vicini & Conti, 1997;D'Sa et al, 2002;Le Jeune et al, 2002).…”

Anti‐inflammatory effects of the novel inhaled phosphodiesterase type 4 inhibitor CHF6001 on virus‐inducible cytokines

“…1999; Seybold et al. 1998). PDE4 inhibitors have proven potential as anti‐inflammatory drugs in chronic airway diseases.…”