Rolipram, salbutamol and prostaglandin E2 suppress TNFα release from human monocytes by activating Type II cAMP-dependent protein kinase

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Abstract

The extent to which cAMP-dependent protein kinase (PKA) mediates the inhibitory effects of cAMP-elevating drugs on tumour necrosis factor (TNF) α release from lipopolysaccharide (LPS)-stimulated human monocytes is equivocal. Here, we have investigated the role of this kinase by exploiting the ability of certain novel cAMP analogues to inhibit or activate PKA and the recently described cAMP-guanine nucleotide-exchange factors (GEFs). Pre-treatment of monocytes with Rp-8-Br-cAMPS, a selective inhibitor of Type I PKA that has no effect on basal or stimulated Rap1 (a downstream effector of cAMP-GEFs) activity, potentiated LPS-induced TNFα output but had little or no effect on the suppression of this cytokine effected by rolipram (a PDE4 inhibitor), prostaglandin (PG) E2 and salbutamol (a β2-adrenoceptor agonist). In contrast, Rp-8-pCPT-cAMPS, which selectively blocks Type II PKA with only weak activity against Rap1, significantly antagonised or abolished the inhibitory effect of these cAMP-elevating agents. Pre-treatment of monocytes with 8-pCPT-2′-O-Me-cAMPS, a potent activator of cAMP-GEFs, failed to suppress TNFα output at concentrations known to profoundly activate Rap1. Collectively, these results indicate that cAMP-elevating drugs suppress TNFα release from LPS-stimulated human monocytes by activating PKA independently of cAMP-GEFs. Furthermore, by using phosphorothioate cAMP analogue PKA inhibitors we provide evidence that the Type II PKA isoenzyme is functionally the most important.

Introduction

cAMP is a classical second messenger and through changes in its rate of synthesis and degradation mediates the effect of a large number of hormones, autacoids and neurotransmitters thereby modulating processes as functionally diverse as visual phototransduction and anaesthesia. Current dogma holds that agonism of Gs-coupled receptors (GsCRs) results in the liberation of Gα from the αβγ heterotrimeric stimulatory guanine nucleotide-binding protein Gs, which augments the basal activity of one or more isoforms of adenylyl cyclase. The cAMP signal then is propagated and amplified down the cAMP-dependent protein kinase (PKA) cascade ultimately to effect a change in cell function [1]. In the inactive state, PKA is tetrameric composed of two identical catalytic (C) subunits, of which at least three subtypes (Cα, Cβ, Cγ) have been identified [2], [3], [4], and two regulatory (R) subunits (RI, RII) that each exist as α and β isoforms [5], [6], [7]. It is the R subunits, RI and RII that provide the basis for the classification of PKA isoenzymes, which are denoted Type I and Type II PKA, respectively. RI and RII feature two, in-tandem, cAMP binding sites designated AI and BI, and AII and BII, respectively [8], [9]. cAMP, when elevated, binds to the R subunits and promotes, in the presence of substrate, the partial or complete dissociation of the inactive holoenzyme thereby releasing free monomeric C subunits [10].

Although much data support the idea that cAMP-elevating drugs act through PKA, there is abundant literature that is inconsistent with this hypothesis [11], [12], [13], [14], [15], [16]. Indeed, it is now known that cAMP can interact with, and/or signal through, multiple intermediates including cGMP-dependent protein kinase (PKG), guanine nucleotide-exchange factors (cAMP-GEFs) and certain ion channels [1], [17]. Thus, contrary to classical doctrine, cAMP-induced responses are not invariably mediated by a common mechanism involving PKA [18].

A significant factor that hampers the unequivocal assignation of PKA to biological responses is a lack of selective pharmacological tools. Many reported compounds marketed as PKA inhibitors are isoquinolinesulfonamides (e.g. H-7, H-8, H-89) or bisindoylmaleimides (e.g. KT 5720) which, in cell-free systems, are non-selective (see http://www.biolog.de/ti1003.html, [19]). Indeed, a recent systematic study of a variety of different protein kinase inhibitors found that H-89 and KT 5720 interacted with many protein kinases and some of these were inhibited more potently than PKA [19].

One approach to circumvent the limitations of currently available, small molecule PKA inhibitors is to deliver to cells the α-isoform of PKA inhibitor (PKIα) by adenovirus vectors [20], [21]. PKIα is a potent and very selective inhibitor of PKA [22], [23], [24] and does not affect the activity of the highly homologous PKG [25] to which it is most closely related [26]. Accordingly, PKIα can be used to assess unequivocally the role of PKA in biological responses [21], [27]. However, adenovirus vectors can only deliver efficiently genes to cells that feature the coxsackie B virus receptor and αv integrins (essential for attachment and internalisation of adenovirus, respectively [28]). Indeed, it is a lack of these ‘receptors’ that renders many cells including lymphocytes, neutrophils, eosinophils and cells of the monocytes/macrophage lineage notoriously resistant to infection. An alternative method to inhibit PKA in adenovirus-resistant cells is to use certain metabolically-stable, phosphorothioate cAMP analogues that can discriminate between the binding sites on RI and RII at concentrations that have weaker effects on other cAMP-binding proteins such as cAMP GEFs [29], [30], [31]. Given the controversy that surrounds the mechanism of action of cAMP-elevating in many pro-inflammatory and immune cells [11], [13], [14], [15], [16] we have exploited the antagonist action of Rp-cAMPS analogues to test the hypothesis that PKA mediates the inhibitory effects of a variety of cAMP-elevating drugs on TNFα release from human monocytes.

Section snippets

Isolation and purification of human mononuclear cells

Blood was collected from normal healthy individuals by ante-cubital venepuncture into acid citrate dextrose (in mM: disodium citrate 160, glucose 110—pH 7.4) and mixed with 6% w/v Hespan (hydroxymethyl starch) to sediment erythrocytes. After standing at room temperature for 90 min, the leukocyte-rich plasma was removed and centrifuged at 312g for 7 min. The resulting cell pellet was resuspended gently in approximately 7 ml of buffer A (in mM: KH2PO4 5, K2HPO4 5, NaCl 110—pH 7.4) made 50% (v v−1)

Expression of C and R subunits in monocytes

Western blotting with rabbit polyclonal antibodies was used to determine the complement of C and R subunits expressed in resting human monocytes. As shown in Fig. 1a, the anti-Cα and anti-Cβ antibodies labelled peptides in human monocytes that migrated, respectively as 40 and 46 kDa bands on SDS–polyacrylamide gels and were identical in size to Cα and Cβ expressed by NIH 3T3 fibroblasts transfected with pCαEV and pCβEV expression vectors [32], [33]. The expression of the Cγ subunit is believed

Discussion

A substantial body of data support the hypothesis that cAMP can suppress the activity of human pro-inflammatory and immune cells. However, whether PKA mediates all of these cAMP-induced responses is equivocal [11], [12], [13], [14], [15], [16]. One controversial example is the negative regulation by cAMP-elevating drugs of TNFα release from LPS-stimulated human monocytes. In these cells, PGE2 and β2-adrenoceptor agonists suppress TNFα generation whereas forskolin, even at high concentrations,

Acknowledgements

This work was supported by the National Asthma Campaign (UK). M.A.G. is an Alberta Heritage Foundation Senior Medical Scholar and is funded by the Canadian Institutes of Health Research.

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