The effects of H89, a PKA inhibitor, on ß2-adrenoceptor stimulated cre-mediated gene transcription responses in CHO-K1 cells J.G.Baker, I.P.Hall & S.J.Hill. Institute of Cell Signalling, Queen's Medical Centre, Nottingham NG7 2UH, UK.

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H89 is a well known PKA inhibitor that has also been reported to have ß-adrenoceptor antagonist properties (Penn et al., 1999). We have previously shown in CHO cells that agonist induced CRE-mediated gene transcription is very sensitive to P42/44 MAP Kinase inhibitors (Baker et al., 2003). The aims of this study were to further investigate the role of cAMP in CRE-mediated gene transcription and examine the effect of H89 on CRE-mediated gene transcription.

CHO-K1 cells stably expressing the human ß2-adrenoceptor and a CRE-SPAP reporter gene were used (McDonnell et al., 1998). Measurements of ³H-CGP 12177 whole cell binding, ³H-cAMP accumulation and CRE-SPAP gene transcription were measured as previously described (Baker et al., 2002).

The cAMP analogues cAMP-8-bromo (1mM) and cAMP-CPT (1mM) and the phosphodiesterase inhibitor IBMX (3mM) all stimulated increases in CRE-SPAP production that were 76.9 + 3.4% (n=3), 85.2 + 4.8% (n=3) and 63.7 + 4.2% (n=5) of the maximum isoprenaline response. These responses were inhibited by 30µM U0126, a MEK1/2 inhibitor to 36.9 + 1.2, 24.9 + 1.5 and 23.7 + 1.5% of the control responses to cAMP-8-bromo, cAMP-CPT and IBMX (n=3, 3, 5) respectively.

In whole cell binding studies, H89 inhibited the binding of ³H-CGP 12177 (log K_D -6.21+0.03, n=4). H89 stimulated an increase in ³H-cAMP accumulation (logEC₅₀ -5.66+0.06, 5.03 +0.8% of the isoprenaline max, n=4). This cAMP response was inhibited by the selective ß2-antagonist ICI 118551 (logK_D -8.83+0.07, n=3). H89 stimulated an increase in CRE-SPAP production (log EC₅₀)-6.51 + 0.05,28.34 ± 1.51% of the maximum isoprenaline response, n=4). This H89 response was also inhibited by ICI 118551 (n=4). No H89 responses were seen in control cells containing the reporter construct, but not the receptor (n=4). The PKA-inhibitory effect of H89 on CRE-gene transcription however was also seen. Pre-incubation with 10µM H89 substantially reduced the forskolin-stimulated CRE-SPAP response (log EC₅₀ -6.40 + 0.06, 91.4 + 2.8% isoprenaline maximum) to 51.08 + 2.77% of control forskolin (n=4) suggesting that part of the CRE-SPAP response is PKA sensitive. On the isoprenaline-stimulated response (log EC₅₀ -9.09 + 0.09, 5.9 + 0.5 fold over basal, n=4), H89 behaved as a partial agonist, shifting the isoprenaline curve to the right (log EC₅₀ in presence of H89 -8.20 + 0.11, n=4) and reducing the maximum response (to 58.1 + 1.2% of control, n=4) in keeping with both its partial ß-agonist and PKA inhibitory effects.

In summary, an increase in intracellular cAMP stimulates CRE-mediated gene transcription. These responses were sensitive to P42/44 MAP Kinase inhibition suggesting a divergent pathway downstream of cAMP. H89 acted as a PKA inhibitor by reducing maximum CRE-gene transcription responses, but also stimulated an increase in both ß2-mediated cAMP accumulation and CRE-SPAP production. Thus H89 was shown to stimulate CRE-mediated gene transcription whilst inhibiting PKA, and hence demonstrating that CRE-gene transcription can occur via a non-PKA dependent route.

JGB holds a Wellcome Trust Clinical Training Fellowship.

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